Pharmaceutical composition for treating bone diseases which comprises protein comprising frizzled1, frizzled2 or frizzled7 extracellular cysteine-rich domain

ABSTRACT

This invention relates to a pharmaceutical composition for treatment of a bone disease comprising, as an active ingredient, a protein comprising an extracellular cysteine-rich domain, which is from the Frizzled receptor selected from the group consisting of mammalian animal-derived Frizzled 1, Frizzled 2, and Frizzled 7 and has activity of increasing bone mass, bone density, and/or bone strength, or a mutant of such domain having sequence identity of 85% or higher to the amino acid sequence of the domain and having activity of increasing bone mass, bone density, and/or bone strength, or a vector comprising a nucleic acid encoding the protein.

TECHNICAL FIELD

The present invention relates to a novel application of a therapeuticagent for a bone disease comprising a protein comprising theextracellular cysteine-rich domain of Frizzled 1, Frizzled 2, orFrizzled 7, which is known as a Wnt ligand receptor protein, or a mutantof the domain.

This finding resulted from analysis of properties of a knock-in mouseexpressing the Frizzled extracellular cysteine-rich domain and a mouseto which a protein comprising the Frizzled extracellular cysteine-richdomain had been administered.

BACKGROUND ART

A super-aging society has arrived, the number of people withosteoporosis has increased, and bone fractures resulting therefrom havecome to constitute a serious issue of concern at a societal level. Inparticular, patients with femoral neck fractures and vertebral bodyfractures become bedridden, which causes significant deterioration ofthe quality of life thereof, and the social, medical, and econimcburdens caused by care and hospital treatment have increased (Tosteson,A. N., et al., Osteoporos Int., 12, 1042-1049, 2001; and Yoh, K., etal., J. Bone Miner. Metab., 23, 167-173, 2005). It has also beendiscovered in recent years that osteoporosis is significantly associatedwith mortality in old age (Nguyen, N. D., et al., J. Bone Miner. Res.,22, 1147-1154, 2007; and Muraki, S., et al., J. Bone Miner. Metab., 24,100-104, 2006). Under such circumstances, prevention and treatment ofosteoporosis have become critical objectives to be achieved.Osteoporosis (i.e., a pathological condition where bone mass is reducedwhile the rate of the amount of the bone matrix to the amount of themineralized bone matrix is held) is classified as primary osteoporosisor secondary osteoporosis. The former type is a pathological conditionheretofore referred to as postmenopausal osteoporosis or senileosteoporosis. The latter type is a pathological condition caused bychanges in bone metabolism resulting from other diseases, and suchosteoporosis is classified based on the cause thereof, such asosteoporosis caused by endocrine, nutritional/metabolic, inflammatory,immobile, drug-induced, hematologic, congenital, or other diseases.According to the above classification, examples of causes for secondaryosteoporosis include: endocrine causes, such as hyperparathyreosis,hyperthyreosis, hypogonadism, Cushing's syndrome, somatotropindeficiency, diabetes, Addison's disease, and calcitonin deficiency;nutritional/metabolic causes, such as chronic degenerative diseases,emaciation, serious liver diseases (primary biliary cirrhosis, inparticular), gastric resection, scorbutus, malabsorption syndrome(including celiac disease), hypophosphatemia, chronic renal disease,hypercalciuria, hemochromatosis, amyloidosis, mast cell tumor, ingestionof excess sodium, insufficient calcium intake, and hypervitaminosis D orA; inflammatory causes, such as articular rheumatism, periarticular bonedisease (elevated bone resorption induced by proinflammatory cytokines),and sarcoidosis; immobility-related causes, such as systemic, bed rest,paralysis, local, and post-fracture causes; drug-induced causes, such aswith the use of steroids (steroids are extensively used for inflammatorydiseases as immunosuppressive agents; examples of diseases treated withthe use of steroids include collagen diseases, asthma, inflammatorybowel diseases, and in the case of organ transplantation, and bone lossis a serious side effect of such therapy), methotrexate, heparine,warfarin, anticonvulsant agents, lithium, and tamoxifen;blood-disease-induced causes, such as multiple myeloma, lymphoma,leukaemia, hemophilia, and chronic hemolytic diseases; congenitalcauses, such as dysosteogenesis, Marfan's syndrome, Kleinfelter'ssyndrome, congenital erythropoetic porphyria, and cystic fibrosis; andother disease-induced causes, such as with chronic obstructive lungdiseases, hepatic failure, renal diseases, articular rheumatism,pregnancy, hyperoxemia, and HIV infection (Committee for Creation ofGuidelines for Prevention and Treatment of Osteoporosis, Guidelines forPrevention and Treatment of Osteoporosis 2006, Life Science Publishing,Co., Ltd., Japan, 2006).

Among the above-mentioned diseases, bone diseases resulting fromosteoarthritis, articular rheumatism, malignant tumors, or renaldiseases are specifically regarded as bone diseases that impose seriousinfluences at the societal level, in addition to primary osteoporosis.

Osteoarthritis develops most often in locomotor regions. The number ofpatients afflicted therewith is said to be 10,000,000 in Japan, and ithas been deduced that the number of patients will keep increasing as theaging of society advances. Advanced articular disorders are treated viaartificial joint replacement; however, radical treatment of moderate ormilder symptoms has not yet been reported (Nampei, A. & Hashimoto, J.,The Bone, 22, 3, 109-113, 2008).

Articular rheumatism is a chronic and progressive inflammatory diseasecharacterized mainly by multiple arthritis. Articular synovialproliferation gradually causes infiltration of cartilage or bones in thevicinity thereof, and articular rheumatism often leads to destructionand deformation of joints. It has been reported that treatment with theuse of an antirheumatic drug (methotrexate) cannot sufficiently inhibitthe progress of joint destruction, and a biological agent targeting atumor necrosis factor (TNF) a produces significant effects of inhibitingjoint destruction. Thus, it is considered to be a revolutionary agent.However, increased incidence, as a side effect, of opportunisticinfection, tuberculosis (extrapulmonary tuberculosis), Pneumocystispneumonia, or the like when using such agent is an issue of concern(Soen, S., The Bone, 22, 3, 103-107, 2008).

Major examples of bone diseases involved in malignant tumors includehypercalcemia and bone metastasis related to malignant tumors.Hypercalcemia causes loss of appetite and diuresis, and it causesdehydration and renal failure caused thereby. Bone metastasis is oftenobserved in patients with breast cancer, prostate cancer, or lungcancer, in particular. While bone metastasis is hardly ever fatal byitself, it causes bone ache, pathologic fracture, neuroparalysis, or thelike. It thus often significantly deteriorate patients' QOL, and bonemetastasis control is a critical objective in clinical settings(Takahashi, S., The Bone, 22, 3, 115-120, 2008). These bone diseasesrelated to malignant tumors are treated with the use of bisphosphonatepreparations, although the problem of side effects has been pointed out.

Among bone diseases related to renal diseases, a pathological conditionof bone damage caused by renal tissue damage is referred to as renalosteodystrophy. Bone disease experienced by kidney dialysis patients aremainly caused by secondary hyperparathyreosis. Because of the elevatedPTH concentration caused by hyperparathyreosis and, for example,insufficient production of bone morphogenetic protein (BMP) 7, renalosteodystrophy advances. Dialysis patients often exhibit loweredreactivity of the bone with the parathyroid hormone (PTH). When the PTHconcentration is chronically and significantly elevated, accordingly,fibrous ostitis (high bone turnover) develops. When the PTHconcentration is maintained within a standard range, in contrast, boneaplasia (low bone turnover) develops.

When fibrous ostitis advances, collagen fibers are irregularly formed,such fibers are mineralized as non-crystalline calcium phosphate, andwoven bone is then formed. This enhances bone formation, although thebone becomes easily fracturable. Basic treatment of fibrous ostitisinvolves inhibition of parathyroid hormone secretion, which mainlyentails calcium ingestion and administration of active vitamin D. When apatient has a chronic kidney disease (CKD) and receives dialysistreatment, in particular, various regulations, such as restrictions onfood or water intake, are necessary. When secondary hyperparathyreosisadvances, hypercalcemia also becomes an issue of concern. Whenprescribing active vitamin D, extreme caution, such as via themonitoring of renal functions (i.e., serum creatinine level) and serumcalcium level, is always required.

Bone aplasia develops because of prolonged use and excessiveadministration of active vitamin D preparations or suppression ofparathyroid hormone after parathyroidectomy (PTX).

The rate of fractures associated with bone aplasia is higher than thatassociated with fibrous ostitis, and it induces hypercalcemia ormineralization of blood vessels or other soft tissues. Thus, adequatetreatment techniques have been desired. A pathological condition of boneaplasia is low bone turnover in which bone resorption and bone formationare inhibited, and there is no established treatment technique atpresent (Daugirdas, J. T., et al., Rinsho Toseki Handbook (Handbook ofDialysis), Fourth Edition, Medical Sciences International, Ltd., Japan,2009).

Hyperphosphatemia or hypercalcemia caused by lowered capacity of thebone for phosphorus or calcium intake (low-turnover metabolic bone) orlowered storage capacity (high-turnover metabolic bone) is considered tobe a cause of ectopic (vascular) mineralization. Cardiovascularcomplications account for 40% or more of the deaths of patients withchronic renal failures, and dialysis patients in particular, andarteriosclerosis involving vascular mineralization has drawn attentionas a serious pathological condition. Treatment of mineralization ofadvanced lesions in patients with chronic renal failures remainsdifficult at present and the prognosis thereof is poor (Fujiu, A. etal., Rinsho Toseki (the Japanese Journal of Clinical Dialysis), 24,43-50, Nihon Medical Center, Japan, 2008). In addition to agents fortreating primary osteoporosis, accordingly, development of agents thatmore effectively act on bone diseases resulting from osteoarthritis,articular rheumatism, malignant tumors, or renal disease and vascularmineralization resulting from bone diseases with reduced side effectshas been desired.

It is considered that bone metabolism is regulated by the balancebetween osteoblast functions and osteoclast functions, and osteoporosisdevelops when the bone-destroying activity exceeds bone-buildingactivity (Cohen, M. M. Jr., American J. Med. Genetics, Part A, 140A,2646-2706, 2006). In particular, secretion of the female hormone thatassumes the role of protecting bones is lowered in postmenopausal women,a lowered capacity of osteoblasts for bone formation and the elevatedbone resorption activity of osteoclasts are consequently observed, andit is highly likely that symptoms of osteoporosis would develop(Kousteni, S., et al., Cell, 104, 719-730, 2001; and Nakamura, T., etal., Cell, 130, 811-823, 2007). In order to overcome such problems,estrogen preparations have been used; however, application thereof hasbeen restricted due to the increased risk of thrombosis and breastcancer caused by the use of such preparations. It is also reported thatuse of a selective estrogen receptor modulator would increase the riskof deep vein thrombosis (Wada, S., et al., Mebio, 25, 8, 89-95, 2008).

At present, calcitonin, bisphosphonate, and the like are used as agentsthat inhibit the bone resorption activity of osteoclasts. Calcitonin isknown to bind to a calcitonin receptor expressed on the osteoclastsurface to inactivate osteoclasts, and it is used for treatment of notonly osteoporosis but also hypercalcemia, Paget's disease of bone, andthe like in clinical settings. However, no effects thereof on bonefracture inhibition have yet been found, and calcitonin receptorexpression is reported to be down-regulated by calcitonin administration(Wada, S., et al., Mebio, 25, 8, 89-95, 2008; and Wada, S. & Yasuda, S.,Clin. Calcium, 11, 9, 1169-1175, 2001). Bisphosphonate exhibits potentbone resorption inhibitory activity, and amino-containingbisphosphonates, such as andronate and risedronate, are majortherapeutic agents for osteoporosis in Japan. Such bisphosphonatepreparations inhibit farnesyl diphosphate synthase, block lipid proteinprenylation, and induce inhibition of bone-resorption functions andosteoclast apoptosis (Nakamura, T., The Bone, 22, 3, 147-151, 2008).However, the FDA warned of crises of severe skeletal, articular, ormuscular pain in 2008 as problems of bisphosphonate preparations. Inaddition, side effects, such as jaw bone necrosis, caused by theprolonged use thereof (i.e., for 2 or 3 years or longer) after dentalcare have been reported (Sanna, G., et al., Ann. Oncol., 16, 1207-1208,2005). An anti-RANKL antibody has been expected as a novel osteoclasticinhibitor other than those described above. Further, application of theanti-RANKL antibody as an inhibitor of articular destruction in the caseof articular rheumatism or as a therapeutic agent for multiple myelomahas been expected, and clinical development thereof is in progress.Based on a report to the effect that the RANKL/RANK pathway is importantfor the survival and maintenance of dendritic cells (Theill, L. E., etal., Ann. Rev. Immunol., 20, 795-823, 2002) or a report to the effectthat lymph node dysplasia is caused in an RANK- or RANKL-deficient mouse(Kong, Y. Y., et al., Nature, 397, 315-323, 1999; and Dougall, W. C., etal., Genes Dev., 13, 2412-2424, 1999), the influence of an anti-RANKLantibody preparation on the immune system has become an issue ofconcern. In 2008, AMGEN reported that an increased rate of developmentof some infectious diseases was found through a clincal test of theanti-RANKL antibody preparation (Denosumab). As a result of the clinicaltest of the anti-RANKL antibody conducted in 2009, development of jawbone necrosis was found to be a side effect, as in the cases of thebisphosphonate preparations. Treatment via intermittent administrationof PTH alone as an osteogenesis accelerator that activates osteoblastshas been conducted (Teriparatide, Eli Lilly; an unapproved drug inJapan), but such agent is not different from other therapeutic agents,such as bisphosphonate preparations, in that activity of increasingcortical bone thickness is not very high compared with activity ofincreasing cancellous bone mass. Accordingly, the effects thereof forbone fracture prevention are not considered to be very high. In relationto PTH, further, Asahi Kasei Pharma Corp. (Japan) has reported problems,such as side effects such as palpitation, tachycardia, and a lowering inblood pressure, and osteosarcoma observed in a long-term administrationtest to rats, unapproved continuous use thereof for 1.5 to 2 years orlonger in Europe and the United States, and prohibited applicationthereof to cancer patients. Thus, it is impossible to use PTH forinhibition of cancer bone metastasis, treatment of cancer-inducedhypercalcemia (paraneoplastic humoral hypercalcemia or local osteolytichypercalcemia caused by the parathyroid-hormone-related peptide producedby tumor cells), or other purposes.

Accordingly, development of agents that more effectively work forosteoporosis caused by the lowered capacity of osteoblasts for boneformation or elevated bone resorption activity of osteoclasts inpostmenopausal women, hypercalcemia, Paget's disease of bone, inhibitionof bone metastasis inhibition of articular destruction associated witharticular rheumatism, or multiple myeloma with reduced side effects hasbeen awaited.

In addition thereto, osteohalisteresis and rachitis are known as bonediseases induced by selective inhibition of mineralization, unlikeosteoporosis. A bone is formed by mineralization of a matrix layercomprising collagen or the like via hydroxyapatite deposition.Osteohalisteresis is a pathological condition in which suchmineralization is blocked and osteoids increase, and it is referred toas rachitis if developed during childhood. Symptoms include bone andjoint pains, such as chiropodalgia, arthralgia, lumbago, and backache,which lead to gait impairment and to a state in which bone is easilyfractured. In the case of children, developmental disorders, limbdeformities such as bow-legs, pigeon breast deformity, or other symptomsare observed. Such symptoms are generally treated with the use ofvitamin D, calcium preparations, and phosphorus preparations, inaddition to alimentary therapy. If the level of dysfunction caused by adeformity is high, however, surgical operation is the only possiblesymptomatic treatment. Therefore, development of agents that are moreeffective on osteohalisteresis or rachitis has been awaited.

As described above, bone is tissue that is always regulated by thebalance between osteoblast functions and osteoclast functions andremodeled. In order to achieve tough bone that is more resistant tofracture, accordingly, a mere increase in bone mass may not besufficient. In the case of hereditary diseases, such as osteopetrosis(Horiuchi A., CLINICIAN, 47, 401-404, 2000), Paget's disease of bone(Daroszewska, A., & Ralston, S. H., Nature Clinical PracticeRheumatology, 2, 270-277, 2006), or Camurati-engelmann's disease (CED)(Janssens, K., et al., Nature Genetics, 26, 273-275, 2000; and Tang, Y.,et al., Nature Medicine, 15, 757-765, 2009), for example, it is knownthat the balance between bone formation and bone resorption becomesabnormal due to different causes, and bone strength is lowered eventhough bone mass is increased. Examples of factors that determine bonestrength from the viewpoint of mechanisms of materials includeform-related factors, such as connectivity of cancellous bones,thickness of cortical bones, porosity, and cross-sectional moment, andqualitative factors, such as mineralization or bone fatigue, in additionto quantitative factors represented by bone density (Mori S., CLINICIAN,49, 621-626, 2002). Therefore, development of agents useful forimproving bone strength, in addition to increasing bone mass, has beenawaited for the purpose of treatment of primary osteoporosis andsecondary osteoporosis.

In recent years, factors associated with the Wnt/LRP signal controlmechanism have drawn attention as targets for drug discovery regarding abone formation accelerator. Wnt is a secreted glycoprotein that has beenlipid-modified by palmitic acid having a molecular weight of about40,000, and 19 types thereof are considered to be present in mammaliananimals. As Wnt receptors, 10 types of seven-transmembrane receptors(i.e., Frizzled receptors) and two types of single-transmembranereceptors (i.e., LRP5/6 receptors) have been reported (Tamai, K., etal., Nature, 407, 530-535, 2000). A region referred to as acysteine-rich domain (CRD) containing conserved 10 cysteine residues ispresent in an extracellular region of the Frizzled receptor familymolecule to which Wnt is considered to bind. The region from thecysteine residue located closest to the N-terminus to the cysteineresidue located closest to the C-terminus of such 10 cysteine residuesmay be exclusively designated as a CRD (Masiakowski, P. & Yancopoulos,G. D., Curr. Biol. 8, R407, 1998), or a region comprising such 10cysteine residues and sequences each located closer to the C- orN-terminus may be designated as a CRD (R & D systems). CRDs werereported to have homodimer structures based on crystal structuralanalysis using a CRD of mouse Frizzled 8 (Dann, C. E., et al., Nature,412, 86-90, 2001). At least three types of Wnt signaling pathways areconsidered to exist: a canonical-Wnt signaling pathway; a non-canonicalWnt signaling pathway, which is a PCP (planar cell polarity) pathwaymediated by a small G-binding protein; and a Ca²⁺ pathway mediated by atrimeric G protein. Bone-metabolism-related research on thecanonical-Wnt signaling pathway is the most advanced, and Wnt isconsidered to promote bone formation (Rawadi, G. & Roman-Roman, S.,Expert Opin. Ther. Targets, 9, 5, 1063-1077, 2005). Therefore,regulation of functions of endogenous factors that inhibit thissignaling pathway has been attempted in recent years for the purpose ofapplication thereof to treatment of bone diseases.

Sclerostin was recognized as a BMP antagonist at first; however, it wasreported to be a factor that would directly bind to LRP5/6 to inhibitthe signaling pathway in research conducted later (Semenov, M., et al.,J. B. C., 280, 29, 26770-26775, 2005). A significant increase wasobserved in bone density in a Sclerostin-knockout mouse (Li, X., et al.,J. Bone Miner. Res., 23, 860-869, 2008). At present, aSclerostin-neutralizing antibody is undergoing phase I trials in Europeand the United States of America (AMG785, Amgen & UCB), and the futuredevelopment thereof has drawn attention. A DKK1(Dickkopf-1)-neutralizing antibody that is known as anothercanonical-Wnt signal inhibitor was prepared, inhibition of lowered bonedensity was observed in an SCID mouse into which multiple myeloma (MM)cells had been transplanted (Yaccoby, S., Blood., 109, 2106-2111, 2007),and clinical trials using a neutralizing antibody (BHQ880, Novartis)have been conducted.

sFRP (soluble frizzled-related protein) that is considered to be a Wntdecoy receptor and has high amino acid sequence homology to the Frizzledextracellular domain is considered to negatively regulate Wnt signals(Nakanishi, R., et al., J. Bone Miner. Res., 21, 1713-1721, 2006), andan increase in the amount of cancellous bone in the femur of an sFRP1knockout mouse has been reported (Trevant, B., et al., J. Cell. Physiol.217, 113-126, 2008). Under such circumstances, research and developmentrelated to sFRP1 inhibitors have proceeded (Wyeth).

Frizzled 7 has been identified as a receptor that binds to a Wnt ligandand transmits signals thereof (Wang, Y., et al., J. B. C., 271, 8,4468-4476, 1996; and Huang, H-C., & Klein, P. S., Genome Biology, 5,234, 1-7, 2004). The amino acid sequence of the human Frizzled 7extracellular cysteine-rich domain (when a region from the cysteineresidue located closest to the N-terminus to the cysteine residuelocated closest to the C-terminus of such 10 conserved cysteine residuesis exclusively designated as a CRD) is completely identical to that ofthe mouse Frizzled 7 extracellular cysteine-rich domain (i.e., there isno difference between species). Involvement thereof with generation anddifferentiation of individual organisms (Wheeler, G. N., CurrentBiology, 10, 849-852, 2000) and involvement thereof with liver cellmultiplication (Matsumoto, K., et al., Dev. Biol., 319, 2, 234-247,2008) have been reported.

Expression patterns of such molecules have been reported: an expressionpattern localized in the crypt base of the mouse small intestine orlarge intestine (Gregorieff, A., et al., Gastroenterology, 129, 626-638,2005); elevated expression levels in various cancer cells (Katoh, M. &Katoh, M., Int. J. Mol. Med., 19, 529-533, 2007); expression in varioustissues (the brain, eyeball, heart, kidney, liver, lung, or spermary)other than those of the spleen via expression analysis of adultmouse-derived tissues of (Wang, Y., et al., J. B. C., 271, 8, 4468-4476,1996); and expression in tissue (the lung or kidney) other than those ofthe brain and the liver via expression analysis of human fetal tissueand potent expression in the skeletal muscle and relatively potentexpression in the heart, weak expression in the brain, the placenta, andthe kidney; and no expression in the lung, the liver, the pancreas, thespleen, the thymic gland, the prostate, the testicle, the ovary, thesmall intestine, or the large intestine via expression analysis of adulthuman-derived tissue (Sagara, N., et al., B. B. R. C., 252, 117-122,1998).

An extracellular cysteine-rich domain that is a soluble receptor of theFrizzled receptor is considered to bind to Wnt and inhibit functionsthereof. It is reported by an in vitro experimentation system that afusion product of the Frizzled 7 extracellular cysteine-rich domain(comprising a region from the cysteine residue located closest to theN-terminus to the cysteine residue located closest to the C-terminus ofthe conserved 10 cysteine residues and sequences each located closer tothe C- or N-terminus) and Fc (R & D Systems) inhibits stabilization ofcytoplasmic β-catenin by Wnt3a (Kemp, C. R., et al., Dev. Dynanics, 236,2011-2019, 2007). Since the expression level of Frizzled 7 is elevatedin cancer cells, it has drawn attention as a target molecule for tumortreatment (WO 2008/031009; and Merle, P., et al., J. Hepatol., 43, 5,854-862, 2005). Regarding colon cancer cells into which a vector thatexpresses a Frizzled 7 extracellular domain has been introduced, forexample, growth thereof was inhibited to a greater extent in a xenografttumor cell transplantation model compared with colon cancer cells intowhich a control vector had been introduced (Vincan, E., et al.,Differentiation, 73, 142-153, 2005). This suggests the possibility thatFrizzled 7 would be a target of drug discovery for tumor treatment.

As described above, 10 types of Frizzled family molecules have beenreported, and Frizzled 1 and Frizzled 2 have been reported as moleculeshaving particularly high primary sequence homology with Frizzled 7 inthe extracellular cysteine-rich domain (when a region from the cysteineresidue located closest to the N-terminus to the cysteine residuelocated closest to the C-terminus of the conserved 10 cysteine residuesis exclusively designated as a CRD, Daroszewska, A., & Ralston, S. H.,Nature Clinical Practice Rheumatology, 2, 270-277, 2006). The amino acidhomologies of Frizzled 7 in the cysteine rich domain (when a region fromthe cysteine residue located closest to the N-terminus to the cysteineresidue located closest to the C-terminus of the conserved 10 cysteineresidues is exclusively designated as a CRD) of such molecule toFrizzled 1 and Frizzled 2 are 91% and 93% respectively in humans andmice. That is, such amino acid sequence homology is very high. As withthe case of Frizzled 7, Frizzled 1 and Frizzled 2 do not showdifferences between mouse-derived and human-derived amino acid sequencesin the cysteine rich domain (when a region from the cysteine residuelocated closest to the N-terminus to the cysteine residue locatedclosest to the C-terminus of the conserved 10 cysteine residues isexclusively designated as a CRD); i.e., such sequences are 100%consistent with each other.

As with Frizzled 7, it is reported that both Frizzled 1 and Frizzled 2interact with Wnt and Frizzled 1 interacts with Wnt3a to protect thehippocampal neuron from being destroyed by amyloid β peptide (Chacon, M.A., et al., J. Cell Physiol., 217, 215-227, 2008). In addition,regarding Frizzled 1 expression patterns, potent expression in theheart, the placenta, the lung, the kidney, the pancreas, the prostate,and the ovary observed via expression analysis of adult human-derivedtissue and potent expression in the lung and the kidney observed viaexpression analysis of fetus-derived tissue have been reported (Sagara,N., et al., B. B. R. C., 252, 117-122, 1998). Since the expressionlevels of both Frizzled 1 and Frizzled 2 are elevated in the case ofcolon cancer or breast cancer, the correlation thereof with cancerationis suggested, and they have drawn attention as target molecules fortumor treatment (WO 2008/061013; Holcombe, R. F., et al., Mol. Pathol.,55, 220-226, 2002; and Milovanovic, T., et al., Int. J. Oncology, 25,1337-1342, 2004). Further, it was reported that Frizzled 1 would notcause any changes in the phenotype of the Frizzled 1 gene-disruptedmouse (Deltagen, Inc., “NIH initiative supporting placement of Deltagen,Inc. mice into public repositories” MGI Direct Data Submission 2005(http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=alleleDetail&key=40116)).When a protein comprising an extracellular cysteine-rich domain derivedfrom the Frizzled 1, Frizzled 2, or Frizzled 7 receptor is expressed invivo at high levels or when a protein comprising an extracellularcysteine-rich domain derived from Frizzled 1, Frizzled 2, or Frizzled 7is administered in vivo, accordingly, it has been very difficult todeduce that such protein would promotively and specifically function soas to increase bone mass and to enhance bone strength.

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

Since the arrival of a super-aging society, treatment of bone diseasesresulting from osteoporosis, osteoarthritis, articular rheumatism, andmalignant tumors, and bone diseases associated therewith have becomecritical issues in the society, and research and development regardingtherapeutic agents for bone diseases have been extensively and activelyconducted. At present, bisphosphonate is one of the most commonly usedagents, and it has high efficacy, although the side effects thereof havebecome problematic in recent years. Problems to be overcome have beenpointed out regarding other agents. Therefore, development of an agentthat is more effective for treatment of bone diseases with reduced sideeffects has been strongly desired.

Means for Solving the Problem

Despite earlier deductions, surprisingly, the present inventors have nowfound for the first time that, when a protein comprising anextracellular cysteine-rich domain derived from the Frizzled 1, Frizzled2, or Frizzled 7 receptor is expressed in vivo at high level or when aprotein comprising an extracellular cysteine-rich domain derived fromFrizzled 1, Frizzled 2, or Frizzled 7 is administered in vivo, suchprotein would promotively and specifically function so as to increasethe bone mass and to enhance bone strength.

The present inventors have now prepared a mouse overexpressing a fusionprotein, which comprises Frizzled 1, Frizzled 2, or Frizzled 7extracellular cysteine-rich domain, and Fc, and discovered based onoverexpression of the fusion of a protein, which comprises the Frizzled7 extracellular cysteine-rich domain, and Fc the following: whitening ofthe femur, whitening of the sternum, whitening and hardening of thecranium, whitening and hardening of the spondylus, and hardening of thecosta; the increased femoral cancellous bone and the increased sternalcancellous bone via observation of H&E stained pathological sections;increased tibial bone density via X-ray photography; increases in thetibial bone volume/tissue volume, the mineral apposition rate, themineralization surface, and the bone formation rate via bonemorphometry; the increased maximum load of femur via bone strengthassays; the increased bone volume/tissue volume, increased trabecularthickness, increased trabecular number, decreased trabecular separation,or decreased trabecular spacing in the cancellous bone region of theproximal tibial metaphysis or the distal femoral metaphysis via bonestructural analysis; and the increased femoral cancellous bones, thethickened diaphyseal wall, and the increased sternal cancellous bone viaobservation of H&E stained pathological sections. They also discoveredbased on overexpression of the fusion of a protein, which comprises theFrizzled 1 extracellular cysteine-rich domain, and Fc: whitening of thefemur, whitening of the sternum, whitening and hardening of the cranium,hardening of the spondylus, and hardening of the costa; the increasedtibial bone density via X-ray photography; the thickened diaphysealwall, the increased cancellous bone, and the increased sternalcancellous bone of the femur via observation of H&E stained pathologicalsections; increases in the increased tibial bone volume/tissue volume,the mineral apposition rate, the mineralization surface, and the boneformation rate via bone morphometry; the increased maximum load of femurvia bone strength assays; and the increased bone volume/tissue volume,the increased trabecular thickness, the increased trabecular number, thedecreased trabecular separation, and the decreased trabecular spacing inthe cancellous bone region at the distal femoral metaphysis via bonestructural analysis. They further discovered based on overexpression ofthe fusion of a protein, which comprises the Frizzled 2 extracellularcysteine-rich domain, and Fc: whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, hardening of thespondylus, and hardening of the costa; the thickened femoral diaphysealwall via observation of H&E stained pathological sections; increases inthe tibial bone volume/tissuel volume, the mineral apposition rate, themineralization surface, and the bone formation rate via bonemorphometry; the increased maximum load of femur via bone strengthassays; and the increased bone volume/tissue volume, the increasedtrabecular thickness, the increased trabecular number, the decreasedtrabecular separation, and the decreased trabecular spacing in thecancellous bone region at the distal femoral metaphysis via bonestructural analysis.

Further, the present inventors have now obtained a fusion protein, whichcomprises the Frizzled 1, Frizzled 2, or Frizzled 7 extracellularcysteine-rich domain, and Fc, as a recombinant protein. The presentinventors have now discovered via administration of the obtainedrecombinant fusion protein, which comprises the Frizzled 7 extracellularcysteine-rich domain, and Fc to a mouse: whitening of the femur,whitening of the cranium, whitening of the sternum, and a tendency ofthickening node; the thickened femoral diaphyseal wall via observationof H&E stained pathological sections; and increased bone volume/tissuevolume in the secondary cancellous bone at the tibial metaphysis viabone morphometry. The present inventors have also discovered viaadministration of the obtained recombinant fusion protein of a protein,which comprises the Frizzled 7 extracellular cysteine-rich domain:whitening of the femur, whitening of the sternum, whitening andhardening of the cranium, hardening of the costa, and hardening of thespondylus, and Fc to an ovariectomized mouse (OVX); the thickenedfemoral diaphyseal wall via observation of H&E stained pathologicalsections; the increased cortical bone cross-sectional area of the femurvia 2D micro CT; and the increased maximum load of femur via bonestrength assays. Further, the present inventors have now administeredthe obtained recombinant fusion protein of a protein, which comprises aminimum CRD sequence comprising the amino acid sequence from N-terminalside cysteine-1 to C-terminal side cysteine-10 in the Frizzled 7extracellular cysteine-rich domain to a mouse. As a result, the presentinventors have now discovered the increased maximum load of femur viabone strength assays and the increased bone volume/tissue volume, theincreased trabecular thickness, the increased trabecular number, thedecreased trabecular separation, and the decreased trabecular spacing ofthe tibia via bone structural analysis. Also, the present inventors havenow administered the obtained recombinant fusion protein of a protein,which comprises the Frizzled 1 extracellular cysteine-rich domain, andFc to a mouse and observed that whitening and epiphyseal hypertrophy ofthe femur, whitening of the sternum, whitening and hardening of thecranium, and hardening of the costa had occurred.

Based on such findings, it was demonstrated that a therapeutic agent fora bone disease comprising, as an active ingredient, a protein comprisingthe Frizzled 1, Frizzled 2, or Frizzled 7 extracellular cysteine-richdomain or a mutant thereof can be provided as a novel therapeutic agentfor a bone disease resulting from osteoporosis, arthritis, or malignanttumors.

Specifically, the present invention includes the following features.

(1) A pharmaceutical composition for treating a bone disease comprising,as an active ingredient, a protein comprising an extracellularcysteine-rich domain, which is from the Frizzled receptor selected fromthe group consisting of mammalian animal-derived Frizzled 1, Frizzled 2,and Frizzled 7 and has activity of increasing bone mass, bone density,and/or bone strength, or a mutant of such domain having sequenceidentity of 85% or higher to the amino acid sequence of such domain andhaving activity of increasing bone mass, bone density, and/or bonestrength, or a vector comprising a nucleic acid encoding the protein.

(2) The composition according to (1), wherein the protein is a fusionprotein of the extracellular cysteine-rich domain or a mutant thereofand the mammalian animal-derived immunoglobulin Fc protein or a mutantthereof, and the nucleic acid encoding the protein is a nucleic acidencoding such fusion protein.

(3) The composition according to (1) or (2), wherein the protein ischemically modified.

(4) The composition according to (3), wherein the chemical modificationis a binding of one or a plurality of polyethylene glycol molecules.

(5) The composition according to (3), wherein the chemical modificationis a binding of a sugar chain.

(6) The composition according to any of (1) to (5), wherein the proteinis a fragment of an extracellular region protein of the Frizzledreceptor, which fragment comprises the extracellular cysteine-richdomain.

(7) The composition according to any of (1) to (6), wherein the proteinis a recombinant protein.

(8) The composition according to any of (1) to (7), wherein theextracellular cysteine-rich domain comprises an amino acid sequencecomprising at least the amino acid sequence spanning from the 1stcysteine residue on the N-terminal side to the 10th cysteine residue inthe amino acid sequence of the extracellular region protein of theFrizzled receptor.

(9) The composition according to any of (6) to (8), wherein theextracellular region protein comprises the amino acid sequence of SEQ IDNO: 19, 20, 22, 23, or 25.

(10) The composition according to any of (1) to (9), wherein the proteincomprising the extracellular cysteine-rich domain comprises a proteincomprising the amino acid sequence spanning from the 1st cysteineresidue on the N-terminal side to the 10th cystein residue as shown inSEQ ID NO: 21, 24, or 26.

(11) The composition according to any of (1) to (10), wherein thenucleic acid encoding a protein comprising the extracellularcysteine-rich domain comprises a nucleotide sequence as shown in any ofSEQ ID NOs: 44 to 49.

(12). The composition according to any of (2) to (11), wherein the Fcprotein comprises the amino acid sequence of SEQ ID NO: 4.

(13) The composition according to any of (2) to (11), wherein thenucleic acid encoding the Fc protein comprises the nucleotide sequenceof SEQ ID NO: 3.

(14) The composition according to any of (2) to (11), wherein the fusionprotein comprises the amino acid sequence as shown in any of SEQ ID NOs:27 to 31.

(15) The composition according to any of (2) to (11), wherein thenucleic acid encoding the fusion protein comprises the nucleotidesequence as shown in any of SEQ ID NOs: 38 to 43.

(16) The composition according to any of (1) to (15), wherein themammalian animal is a human.

(17) The composition according to any of (1) to (16), wherein the bonedisease is accompanied with lowering of bone mass, bone density, and/orbone strength.

(18) A method for treating a bone disease comprising administering thecomposition according to any of (1) to (17) to a mammalian animal.

(19) The method according to (18), wherein the mammalian animal is ahuman.

(20) The method according to (18), wherein the bone disease isaccompanied with lowering of bone mass, bone density and/or bonestrength.

(21) The method according to any of (18) to (20), wherein thecomposition is simultaneously or continuously administered incombination with another therapeutic agent for bone disease.

Effects of the Invention

The present invention can increase bone mass, bone density, and/or bonestrength. Accordingly, a disease involving lowering of bone mass, bonedensity, and/or bone strength, such as a bone disease resulting fromosteoporosis, osteoarthritis, articular rheumatism, malignant tumors, orother diseases and various bone diseases or disorders associatedtherewith can be treated without causing side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of H&E stained pathological sections obtained fromthe femurs of a 16-week-old USmFZD7crd-hFcm KI chimeric mouse (rightdiagram) and a control mouse (left diagram).

FIG. 2 shows images of H&E stained pathological sections obtained fromthe sternums of a 16-week-old USmFZD7crd-hFcm KI chimeric mouse (rightdiagram) and a control mouse (left diagram).

FIG. 3 shows a X-ray photograph of the tibiae of a 16-week-old female(♀) USmFZD7crd-hFcm KI chimeric mouse (lower portion) and a female (♀)control mouse (upper portion).

FIG. 4 shows a X-ray photograph of the tibiae of a 16-week-old male (♂)USmFZD7crd-hFcm KI chimeric mouse (lower portion) and a male (♂) controlmouse (upper portion).

FIG. 5 shows an image showing the results of Western analysis using theserum obtained from the 16-week-old USmFZD7crd-hFcm KI chimeric mouse:wherein 1266 and 1268 represent serum samples obtained from the controlchimeric mouse; A3, A6, B8, and B17 represent serum samples obtainedfrom the USmFZD7crd-hFcm KI chimeric mouse; and an arrow indicates aposition of a main band specific to the serum sample obtained from theUSmFZD7crd-hFcm KI chimeric mouse.

FIG. 6 shows a X-ray photograph of the tibiae of the 16-week-old female(♀) USmFZD1crd-hFcm KI chimeric mouse (lower portion) and the female (♀)control mouse (upper portion).

FIG. 7 shows a X-ray photograph of the tibiae of the 16-week-old male(♂) USmFZD1crd-hFcm KI chimeric mouse (lower portion) and the male (♂)control mouse (upper portion).

FIG. 8 shows images of H&E stained pathological sections obtained fromthe femoral diaphyses of the 16-week-old USmFZD1crd-hFcm KI chimericmouse (right diagram) and the control mouse (left diagram).

FIG. 9 shows images of H&E stained pathological sections obtained fromthe femurs (at a site 50% away from the proximal end) of the 16-week-oldUSmFZD1crd-hFcm KI chimeric mouse (right diagram) and the control mouse(left diagram).

FIG. 10 shows images of H&E stained pathological sections of theproximal femoral growth plates of the 16-week-old USmFZD1crd-hFcm KIchimeric mouse (right diagram) and the control mouse (left diagram).

FIG. 11 shows images of H&E stained pathological sections of thesternums of the 16-week-old USmFZD1crd-hFcm KI chimeric mouse (rightdiagram) and the control mouse (left diagram).

FIG. 12 shows a recombinant mFZD7crd-hFcm expression vector.

FIG. 13 shows a recombinant mFZD1crd-hFcm expression vector.

FIG. 14 shows images of H&E stained pathological sections of the femurs(at a site 30% away from the proximal end) of the 12-week-oldUShFZD7crd-hFcm KI chimeric mouse (right diagram) and the control mouse(left diagram).

FIG. 15 shows images of H&E stained pathological sections of the femurs(at a site 50% away from the proximal end) of the 12-week-oldUShFZD7crd-hFcm KI chimeric mouse (right diagram) and the control mouse(left diagram).

FIG. 16 shows images of H&E stained pathological sections of the femursof the 12-week-old UShFZD7crd-hFcm KI chimeric mouse (right diagram) andthe control mouse (left diagram).

FIG. 17 shows images of H&E stained pathological sections of thesternums of the 12-week-old UShFZD7crd-hFcm KI chimeric mouse (rightdiagram) and the control mouse (left diagram).

FIG. 18 shows 2D micro CT images of the femoral cortical bone (at a site50% away from the proximal end) of the sham/non-treatment group (upperleft diagram), the OVX/non-treatment group (lower left diagram), thesham/mFZD7crd-hFcm group (upper right diagram), and theOVX/mFZD7crd-hFcm group (lower right diagram).

FIG. 19 shows images of H&E stained pathological sections of the femurs(at a site 30% away from the proximal end) of the 12-week-oldUShFZD1crd-hFcm KI chimeric mouse (right diagram) and the control mouse(left diagram).

FIG. 20 shows images of H&E stained pathological sections of the femurs(at a site 50% away from the proximal end) of the 12-week-oldUShFZD1crd-hFcm KI chimeric mouse (right diagram) and the control mouse(left diagram).

FIG. 21 shows images of H&E stained pathological sections of the femursof the 12-week-old UShFZD1crd-hFcm KI chimeric mouse (right diagram) andthe control mouse (left diagram).

FIG. 22 shows images of H&E stained pathological sections of thesternums of the 12-week-old UShFZD1crd-hFcm KI chimeric mouse (rightdiagram) and the control mouse (left diagram).

FIG. 23 shows a recombinant mFZD2crd-hFcm expression vector.

FIG. 24 shows a recombinant mFZD7c10-hFcm expression vector.

FIG. 25 shows 3D micro CT images of tibial cancellous bones of a mouseto which a recombinant mFZD7c10-hFcm protein has been administered(right diagram) and the control mouse (left diagram).

PREFERRED EMBODIMENTS OF THE INVENTION

Hereafter, the present invention is described in detail.

As described above, the present invention provides a pharmaceuticalcomposition for treatment of a bone disease comprising, as an activeingredient, a protein which comprises an extracellular cysteine-richdomain derived from the Frizzled receptor selected from the groupconsisting of mammalian animal-derived Frizzled 1, Frizzled 2, andFrizzled 7 and having activity of increasing bone mass, bone density,and/or bone strength or a mutant thereof having a 85% or higher sequenceidentity to the amino acid sequence of said domain and having activityof increasing bone mass, bone density, and/or bone strength, or a vectorcomprising a nucleic acid encoding said protein.

The present invention is based on the finding of that a fragmentcomprising an extracellular cysteine-rich domain in the extracellularregion protein of a Frizzled receptor has a function of increasing thebone mass, bone density, and/or bone strength of a mammalian animal.Specifically, the present inventors have now prepared a mouse expressingthe Frizzled extracellular cysteine-rich domain from mouse ES cells by aknock-in technique or administered a recombinant fusion protein of aprotein, which comprises the Frizzled extracellular cysteine-richdomain, and Fc to a mouse. As a result, they have now found, for thefirst time, that the bone mass, bone density, and/or bone strength at abone site of interest would be increased to the extent that suchincrease could be visually and sensuously recognized compared withwild-type mice. Further, the present inventors have now discovered that,surprisingly, the effects of the extracellular cysteine-rich domain forincreasing the bone mass, bone density, and/or bone strength werebone-specific and such effects were not influential at all orsubstantially not influential on other tissue or organs; i.e., no sideeffects were observed. According to the past findings, the extracellularcysteine-rich domain of the Frizzled receptor had been considered tobind to Wnt, which is a ligand of the receptor and associated with bonemorphogenesis, and inhibit the functions of the domain, as described inthe “Background Art” above. Thus, such domain was not deduced to beinvolved in bone growth acceleration. The extracellular cysteine-richdomain of Frizzled 7 is reported to be particularly effective forinhibiting proliferation of tumors such as colon cancer and it has drawnattention as the target of drug discovery for cancer treatment.

Thus, the present inventors have now discovered that the extracellularcysteine-rich domain of a Frizzled receptor has novel useful functionsof specifically and promotively increasing the bone mass, bone density,and/or bone strength. The pharmaceutical composition of the presentinvention can be used for treatment of a bone disease aimed atincreasing the bone mass, bone density, and/or bone strength at a bonesite.

Hereafter, the pharmaceutical composition of the present invention isdescribed in greater detail.

<Extracellular Cysteine-Rich Domain of Frizzled Receptor>

The Frizzled receptor of the present invention is mammaliananimal-derived Frizzled 1, Frizzled 2, or Frizzled 7. Such receptor hasparticularly high identity of the extracellular cysteine-rich domain(hereafter, it is occasionally referred to as “CRD”) among ten types ofFrizzled receptors whose ligands are Wnt. Identity of the amino acidsequences comprising N-terminal side cysteine-1 to C-terminal sidecysteine-10 between CRDs of such receptors is 93% between a CRD ofFrizzled 7 and a CRD of Frizzled 2 and 91% between a CRD of Frizzled 7and a CRD of Frizzled 1, in the case of the human- and mouse-derivedsequences. The amino acid sequence of such region of a human isidentical to that of a mouse and highly conserved across species.Sequence identity between a CRD of any of Frizzled 3 to 6 and 8 to 10and that of Frizzled 7 is as low as 42% to 56%.

Information regarding the amino acid and nucleotide sequences ofFrizzled 1, Frizzled 2, and Frizzled 7 is available from NCBI (U.S.A.).

Frizzled 7 (also referred to as “FZD7”) is isolated from, for example,human, mouse, Rhesus monkey, red junglefowl, zebrafish, or Xenopus, andsequence information is open to the public. In the present invention,the origin of the Frizzled 7 protein or a nucleic acid encoding the sameis not limited, and it is preferably derived from, for example, amammalian animal, such as a primate including a human and a rodentincluding mouse. Sequence information of human- or mouse-derivedFrizzled 7 is registered under, for example, Accession Number:NM_003507.1 or NP_003498.1 in the case of human Frizzled 7, or AccessionNumber: NM_008057.1, NP_032083.1, NM_008057.2, NP_032083.2, NM_008057.3,or NP_032083.3 in the case of mouse Frizzled 7, with the GenBank (NCBI,U.S.A.).

The amino acid sequences of the extracellular region proteins of humanand mouse Frizzled 7 are as follows.

Amino acid sequence of extracellular regionprotein of human Frizzled 7 (SEQ ID NO: 19):QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTA PYLAmino acid sequence of the extracellular regionprotein of mouse Frizzled 7 (SEQ ID NO: 20):QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGAGGSPTAYPTA PYL

The underlined portion represents a sequence comprising N-terminal sidecysteine-1 to C-terminal side cysteine-10, which is the minimal CRDregion (SEQ ID NO: 21).

SEQ ID NO: 21: CQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRC ENFPVHGAGEIC

Frizzled 1 (also referred to as “FZD1”) is isolated from, for example,human, mouse, rat, red junglefowl, or Xenopus, and sequence informationis open to the public. In the present invention, the origin of theFrizzled 1 protein or a nucleic acid encoding the same is not limited,and it is preferably derived from, for example, a mammalian animal, suchas a primate including a human and a rodent including mouse. Sequenceinformation of human- or mouse-derived Frizzled 1 is registered under,for example, Accession Number: NM_003505.1 or NP_003496.1 in the case ofhuman Frizzled 1, or Accession Number: NM_021457.1, NP_067432.1,NM_021457.2, NP_067432.2, or NM_021457.3 in the case of mouse FZD1, withthe GenBank.

The amino acid sequences of the extracellular region proteins of humanand mouse Frizzled 1 are as follows.

Amino acid sequence of extracellular regionprotein of human Frizzled 1 (SEQ ID NO: 22):QAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHAmino acid sequence of extracellular regionprotein of mouse Frizzled 1 (SEQ ID NO: 23):QAAGQVSGPGQQAPPPPQPQQSGQQYNGERGISIPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQH

The underlined portion represents a sequence spanning from the 1stcysteine residue on the N-terminal side to the 10th cystein residue onthe C-terminal side, which portion is the minimal CRD region (SEQ ID NO:24). The amino acid sequence of this region of a human is identical tothat of a mouse.

SEQ ID NO: 24: CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKC EKFPVHGAGELC

Frizzled 2 (also referred to as “FZD2”) is isolated from, for example,human, mouse, rat, or Xenopus, and sequence information is open to thepublic. In the present invention, the origin of the Frizzled 2 proteinor a nucleic acid encoding the same is not limited, and it is preferablyderived from, for example, a mammalian animal, such as a primateincluding a human and a rodent including mouse. Sequence information ofhuman- or mouse-derived Frizzled 2 is registered under, for example,Accession Number: NM_001466.1, NM_001466.2, or NP_001457.1 in the caseof human Frizzled 2, or Accession Number: NM_020510.1, NM_020510.2,NP_065256.1 in the case of mouse FZD2, with the GenBank.

The amino acid sequences of the extracellular region proteins of humanFrizzled 2 is identical to that of mouse Frizzled 2 as shown below.

Amino acid sequences of extracellular regionproteins of human and mouse Frizzled 2 (SEQ ID NO: 25):QFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPAL

The underlined portion represents a sequence spanning from the 1stcysteine residue on the N-terminal side to the 10th cystein residue onthe C-terminal side, which portion is the minimal CRD region (SEQ ID NO:26).

SEQ ID NO: 26: CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRC EHFPRHGAEQIC

In the present invention, the term “extracellular cysteine-rich domain”refers to a protein which comprises at least an amino acid sequencespanning the 1st cysteine residue on the N-terminal side to the 10thcysteine residue in the extracellular region protein of the Frizzledreceptor selected from the group consisting of mammalian animal-derivedFrizzled 1, Frizzled 2, and Frizzled 7 and which is capable ofincreasing the bone mass, bone density and/or bone strength of amammalian animal. The expression “comprising at least” as used hereinmeans that the extracellular cysteine-rich domain may be composed of aminimum CRD sequence spanning from the 1st cysteine residue on theN-terminal side to the 10th cysteine residue in the extracellular regionprotein of the Frizzled receptor, or alternatively that any foreignsequence may be added to the N- and/or C-terminus of the minimum CRDsequence, provided that the resulting sequence has an ability toincrease the bone mass, bone density, and/or bone strength. The term“foreign sequence” may refer to, for example, a sequence derived fromany foreign protein unrelated to the extracellular region protein of theFrizzled receptor, an artificial sequence, or a sequence derived from aportion of the extracellular region protein of a foreign Frizzledreceptor other than the minimum CRD sequence.

The extracellular cysteine-rich domain according to the presentinvention is a protein which comprises an amino acid sequence comprisingat least the amino acid sequence spanning from the 1st cysteine residueon the N-terminal side to the 10th cystein residue in the extracellularregion protein of the Frizzled receptor selected from the groupconsisting of mammalian animal-derived Frizzled 1, Frizzled 2, andFrizzled 7 and which is capable of increasing the bone mass, bonedensity, and/or bone strength of a mammalian animal. The expression“comprising at least” as used herein means that the minimum sequenceconsists of the amino acid sequence spanning the 1st cystein residue onthe N-terminal side to the 10th cysteine residue in the extracellularregion protein of the Frizzled receptor, and a sequence derived from theextracellular region protein of the Frizzled receptor of the samespecies may be adequately extended and comprised at the N-terminusand/or C-terminus of the minimum sequence. Accordingly, theextracellular cysteine-rich domain can comprise any amino acid sequencespanning from the aforementioned minimum CRD sequence to the maximum CRDsequence of the extracellular region protein of the Frizzled receptor.

In the present invention, examples of mammalian animals include, but arenot limited to, primates, livestock animals, rodents, ungulates, and petanimals. Preferable mammalian animals are humans and mice. Mice areimportant since they have the amino acid sequence of the extracellularcysteine-rich domain (CRD); specifically, the minimum CRD sequencespanning from the 1st cystein residue on the N-terminal side to the 10thcystein residue on the C-terminal side is identical to a human-derivedsequence.

In the present invention, a preferable CRD is a protein which comprisesan amino acid sequence comprising at least the amino acid sequencespanning from the 1st cystein residue on the N-terminal side to the 10thcysteine residue in the extracellular region protein of the Frizzledreceptor selected from the group consisting of human- or mouse-derivedFrizzled 7, Frizzled 1, and Frizzled 2 (SEQ ID NO: 21, 24, or 26) andwhich has an ability to increase the bone mass, bone density, and/orbone strength of a mammalian animal.

In the present invention, another preferable CRD is a protein whichcomprises an amino acid sequence comprising at least the amino acidsequence spanning the 1st cystein residue on the N-terminal side to the10th cystein residue (as shown in SEQ ID NO: 21, 24, or 26) in the aminoacid sequence (as shown in SEQ ID NO: 19, 20, 22, 23, or 25,respectively) of the extracellular region protein of the Frizzledreceptor selected from the group consisting of human- or mouse-derivedFrizzled 7, Frizzled 1, and Frizzled 2 and which has an ability toincrease the bone mass, bone density, and/or bone strength of amammalian animal.

In the present invention, an increase in “the bone mass, bone density,and/or bone strength” involves at least the increased cancellous bone,the thickened and proliferated diaphysis, or the increased maximum load,for example.

<Mutant of Extracellular Cysteine-Rich Domain>

The extracellular cysteine-rich domain of the present invention includesa mutant of the extracellular cysteine-rich domain described in thesection of <Extracellular cysteine-rich domain of Frizzled receptor>above. Such mutant may be a naturally-occurring or artificial mutant,which comprises an amino acid sequence comprising a substitution(s),deletion(s), or addition(s) of one or more (preferably one or several)amino acids in the amino acid sequence of the extracellularcysteine-rich domain, or comprises an amino acid sequence having 80% orhigher, preferably 85% or higher, and more preferably 90% or higher,such as 93% or higher, 95% or higher, 97% or higher, 98% or higher, or99% or higher identity with the amino acid sequence of the extracellularcysteine-rich domain, and which has an ability to increase the bonemass, bone density, and/or bone strength.

For example, the mutant comprises an amino acid sequence comprising asubstitution(s), deletion(s), or addition(s) of one or more (preferablyone or several) amino acids in the amino acid sequence as shown in SEQID NO: 21, 24 or 26, 19, 20, 22, 23, or 25, or comprises an amino acidsequence having 80% or higher, preferably 85% or higher, and morepreferably 90% or higher, such as 93% or higher, 95% or higher, 97% orhigher, 98% or higher, or 99% or higher identity with the amino acidsequence as shown in SEQ ID NO: 21, 24 or 26, 19, 20, 22, 23, or 25, andthe mutant has an ability to increase the bone mass, bone density,and/or bone strength.

The term “several” used herein generally refers to an arbitrary integerbetween 2 and 10, and it is preferably an integer between 2 and 5.

The term “identity” as used herein refers to a degree of coincidencebetween two amino acid sequences (or nucleotide sequences) that arealigned to maximize the number of identical amino acid residues (or thenumber of identical nucleotides). Specifically, the identity isrepresented by a percentage (%) of the number of identical amino acidresidues (or the number of identical nucleotides) relative to the totalnumber of amino acid residues (or the total number of nucleotides). Whena gap is introduced as in the case of FASTA, the number of gaps is addedto the total number of amino acid residues (or the total number ofnucleotides).

Proteins having 80% or higher, and preferably 85% or higher sequenceidentity, can be screened for by accessing, for example, the sequencedatabases of NCBI (U.S.A.) or EMBL (Europe) and utilizing a sequencehomology search program, such as BLAST or FASTA (e.g., Altschul, S. F.et al., 1990, J. Mol. Biol. 15:403-410; Karlin, S. and Altschul S. F.,1990, Proc. Natl. Acad. Sci., U.S.A., 87: 2264-2268). According toBLAST, a sequence is divided into words of a fixed length, similarfragments are screened for in the word unit, such fragments are extendedtoward the both directions to maximize the similarity, local alignmentis performed, and the aligned sequences are bound in the end to performthe final alignment. According to FASTA, continuously coincide sequencefragments are screened for at a high speed, fragments exhibiting highsimilarity are selectively subjected to local alignment, the fragmentsare bound to each other in the end while gaps are taken intoconsideration to perform alignment.

When a mutation is introduced into the extracellular cysteine-richdomain of the present invention, it is preferable that amino acidresidues other than 10 cysteine residues in the sequence spanning the1st cystein residue on the N-terminal side to the 10th cysteine residueon the C-terminal side of the extracellular region protein of theFrizzled receptor be exclusively subjected to a mutation ofsubstitution, deletion, or addition, natural disulfide bonds be notdestructed, and a natural conformation be substantially maintained. If anatural disulfide bond(s) in the extracellular cysteine-rich domain isdestructed and an inherent conformation is altered, the protein domainmay disadvantageously lose or significantly reduce the ability ofincreasing the bone mass, bone density, and/or bone strength.

A preferable mutagenesis technique is a site-directed mutagenesisutilizing PCR involving the use of primers synthesized based on theknown sequence of the extracellular cysteine-rich domain (including acomplementary mutant sequence) (e.g., Kunkel et al., Proc. Natl. Acad.Sci., U.S.A., 1985, 82: 488-492; F. M. Ausubel et al., Short Protocolsin Molecular Biology, 1995, John Wiley & Sons; J. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., 1989, Cold SpringHarbor Laboratory Press). Since mutagenesis kits are commerciallyavailable (e.g., Takara Shuzo Co., Ltd.), mutation can be introducedwith the use of such kits in accordance with the instructions.

Briefly, the method of Kunkel comprises using a plasmid containing DNAencoding the extracellular cysteine-rich domain as a template, annealinga primer having a phosphorylated 5′ terminus with T4 DNA polynucleotidekinase (including a complementary mutant sequence) to the template,synthesizing DNA, ligating the terminuses with the aid of T4 DNA ligase,and purifying DNA containing mutation of interest.

In the present invention, the mutation includes a substitution, adeletion, an addition, an insertion, or combinations thereof.

Substitution may be conservative or non-conservative. Conservativesubstitution is preferable in order to substantially refrain fromaltering the conformation of a protein of the extracellularcysteine-rich domain. The term “conservative substitution” refers tosubstitution across amino acids having similar structural properties(e.g., a branch state or aromaticity), electric properties (e.g., acidicor basic properties), and chemical and physical properties (e.g., polaror hydrophobic properties). Examples of branched amino acids includevaline, leucine, and isoleucine. Examples of aromatic amino acidsinclude tyrosine, tryptophan, phenylalanine, and histidine. Examples ofacidic amino acids include glutamic acid and aspartic acid. Examples ofbasic amino acids include lysine, arginine, and histidine. Examples ofpolar amino acids include serine, threonine, glutamine, asparagine,tyrosine, cysteine, glycine, and proline. Examples of hydrophobic aminoacids include alanine, valine, leucine, isoleucine, and methionine.

Deletion involves loss of one or a plurality of amino acid residues.Addition involves binding of one or a plurality of amino acid residuesto the protein N- or C-terminus. Insertion involves binding of one or aplurality of amino acid residues to the inside of a protein. Deletionand insertion can be performed, provided that a protein conformation ofthe extracellular cysteine-rich domain is not substantially changed.Thus, the number of amino acid residues that can be subjected todeletion or insertion is preferably limited to about 1 to 5.

<Protein Comprising an Extracellular Cysteine-Rich Domain or a MutantThereof>

As described above, an active ingredient of the pharmaceuticalcomposition of the present invention is a protein comprising anextracellular cysteine-rich domain derived from the Frizzled receptorselected from the group consisting of mammalian animal-derived Frizzled1, Frizzled 2, and Frizzled 7 and having activity of increasing bonemass, bone density, and/or bone strength or a mutant thereof having 85%or higher sequence identity to the amino acid sequence of such domainand having activity of increasing bone mass, bone density, and/or bonestrength.

The expression “comprise” or “comprising” used herein refers that theextracellular cysteine-rich domain or a mutant thereof may comprise aforeign peptide, polypeptide, or protein bound or fused to the N- orC-terminus of such domain or a mutant thereof via an adequate peptidelinker (e.g., 1 to 20 amino acid residues), where needed. Examples ofpreferable foreign proteins include the mammalian animal-derivedimmunoglobulin Fc protein and a mutant thereof. Since a rejectionreaction may take place upon administration of such foreign protein inan organism, it may be preferable that a protein inherent to a mammaliananimal to which such protein is to be administered be used as theforeign protein, in order to avoid such rejection as much as possible.

A preferable Fc protein is a human immunoglobulin Fc protein from theviewpoint of application thereof to a human. Examples of immunoglobulinclasses and subclasses include, but are not limited to, IgG, IgD, IgE,IgM, IgA, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA1, and IgA2.Use of a human immunoglobulin class and subclass is preferable if theprotein is applied to a human. The Fc protein can improve stability ofthe extracellular cysteine-rich domain or a mutant thereof in vivo. Insuch a case, however, biological activity, such as antibody dependentcellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity(CDC), of the Fc protein is preferably lowered in advance in order toavoid the influence of such biological activity in vivo. To this end, itis preferable that a mutation for suppressing, lowering, or losing suchbiological activity be introduced. Such mutation is amino acidsubstitution of, for example, 1 to 10, preferably 1 to 5, and morepreferably 1 to 3 amino acid residues in the amino acid sequence of themammalian animal-derived Fc protein. Arbitrary amino acid substitutionreduces ADCC and/or CDC activity. A specific example is substitution asdescribed in Example 1 below. A preferable example of the Fe protein isa human IgG1 Fc mutant comprising the amino acid sequence as shown inSEQ ID NO: 4. An Fc protein may bound to an N- or C-terminal site of theextracellular cysteine-rich domain or a mutant thereof, with theC-terminal site being preferable.

Specific examples of the Fc fusion protein include proteins comprisingamino acid sequences as shown in SEQ ID NOs: 27 to 31 below. Theunderlined portion represents a protein comprising the extracellularcysteine-rich domain and the non-underlined portion represents a humanIgG1 Fc mutant protein.

SEQ ID NO: 27 (SEQ ID NO: 19 + SEQ ID NO: 4):QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 28 (SEQ ID NO: 20 +SEQ ID NO: 4): QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGAGGSPTAYPTAPYLAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 29 (SEQ ID NO: 22 +SEQ ID NO: 4): QAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKSEQ ID NO: 30 (SEQ ID NO: 23 + SEQ ID NO: 4):QAAGQVSGPGQQAPPPPQPQQSGQQYNGERGISIPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKSEQ ID NO: 31 (SEQ ID NO: 25 + SEQ ID NO: 4):QFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The extracellular cysteine-rich domain in the amino acid sequence asshown in any of SEQ ID NOs: 27 to 31 is derived from the extracellularregion protein of the Frizzled 7, Frizzled 1, or Frizzled 2 receptor,and the amino acid sequence of such domain may include mutation asdescribed in the above <Mutant of extracellular cysteine-rich domain>,provided that it has the capacity for increasing the bone mass, bonedensity, and/or bone strength.

In the present invention, a protein comprising the extracellularcysteine-rich domain or a mutant thereof is not always required to bindor fuse to a foreign peptide, polypeptide, or protein. Specifically, theprotein of the present invention may be a fragment of the extracellularregion protein of the Frizzled 1, 2, or 7 receptor comprising theaforementioned extracellular cysteine-rich domain. Such fragment mayinclude a mutation as described in the above <Mutant of extracellularcysteine-rich domain>, provided that the mutant has the capacity forincreasing the bone mass, bone density, and/or bone strength.

The protein comprising the extracellular cysteine-rich domain or amutant thereof according to the present invention can be prepared via agene recombination technique common in the art. Briefly, such proteinpreparation comprises preparing DNA encoding the protein of the presentinvention, constructing an expression vector comprising the DNA,transforming or transfecting prokaryotic or eukaryotic cells with theuse of such vector, and recovering a target recombinant protein from thecultured cells. Protein purification can be carried out by employingcommon protein purification techniques, such as ammonium sulfateprecipitation, organic solvent precipitation, dialysis, electrophoresis,chromatofocusing, gel filtration chromatography, ion exchangechromatography, affinity chromatography, and HPLC, in adequatecombination.

The DNA and the vector mentioned above are as described in the above<Nucleic acid and vector> and Examples below. Gene recombinationtechniques described in, for example, F. M. Ausubel et al., ShortProtocols in Molecular Biology, 1995 or John Wiley & Sons, J. Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd ed., 1989, ColdSpring Harbor Laboratory Press can be applied to the present invention.

The protein comprising the extracellular cysteine-rich domain or amutant thereof according to the present invention may be chemicallymodified.

Examples of chemical modification techniques include, but are notlimited to, glycosylation, pegylation (PEG), acetylation, amidation, andphosphorylation. Particularly preferable chemical modificationtechniques are glycosylation and pegylation.

The term “pegylation” refers to binding of one or a plurality ofpolyethylene glycol (PEG) molecules to, for example, an amino acidresidue, such as an N-terminal amino group of a protein or a ε-aminogroup of lysine (Lys). In general, a PEG molecule is bound to a freeamino group of an amino acid. An average molecular weight of PEG can bein the range of, but is not limited to, about 3,000 to about 50,000. PEGcan be bound to a protein by introducing an active group, such as acarboxyl, formyl (aldehyde), N-hydroxysuccinimide ester, amino, thiol,or maleimide group, to a terminus of PEG and allowing such group toreact with a group of a protein, such as an amino, carboxyl, thiol, orhydroxyl group.

The term “glycosylation” refers to binding of a carbohydrate chain(i.e., a sugar chain) to an asparagine, serine, or threonine residue ofa protein. In general, glycosylation takes place upon recognition of anAsn-X-Thr/Ser sequence (wherein X represents an amino acid residue otherthan Pro). When an amino acid sequence of the protein is modified so asto have such sequence, a sugar chain can be introduced into a site thatis different from that of a naturally-occurring protein. In general, anucleic acid encoding a recombinant protein is expressed in aneukaryotic cell (e.g., an yeast, animal, or plant cell) via geneticrecombination to cause glycosylation of a recombinant protein. In thepresent invention, a sugar chain structure is not particularly limited,and it is considered to differ depending on a type of a cell selectedfor expression. When used for a human, a human-derived cell, an yeastcell capable of synthesizing a human sugar chain, a Chinese hamsterovary (CHO) cell, or the like can be used.

It is preferable that acetylation or amidation be mainly carried out atthe protein N- or C-terminus. Such reaction can be carried out with theuse of, for example, an alcohol, such as aliphatic alcohol or fattyacid, or a carboxylic acid. The number of carbon atoms in the alkylmoiety is, for example, about 1 to 20; however, conditions in terms ofwater-solubility and avirulence need to be satisfied.

<Nucleic Acid and Vector>

An example of an active ingredient of the composition of the presentinvention is a vector comprising a nucleic acid encoding a proteincomprising the extracellular cysteine-rich domain or a mutant thereof.

The term “nucleic acid” used herein refers to both DNA and RNA, whereinDNA encompasses genomic DNA and cDNA, and RNA encompasses mRNA.

The extracellular cysteine-rich domain, the mutant thereof, and theprotein comprising the same, including the fusion protein with the Fcprotein, are as described in the above sections <Extracellularcysteine-rich domain of Frizzled receptor>, <Mutant of extracellularcysteine-rich domain>, and <Protein comprising an extracellularcysteine-rich domain or a mutant thereof, and all descriptions made insuch sections are employed herein. Accordingly, the term “nucleic acid”used in the present invention encompasses the nucleic acid encoding aprotein comprising the extracellular cysteine-rich domain or a mutantthereof specifically described above.

Specifically, examples of such nucleic acids include nucleic acidsencoding amino acid sequences comprising at least CRD minimal sequencescomprising amino acid sequences composed of a region from N-terminalside cysteine-1 to C-terminal side cysteine-10 (SEQ ID NOs: 21, 24, and26) derived from the amino acid sequences comprising the extracellularregion protein of mouse Frizzled 7, Frizzled 1, and Frizzled 2 (SEQ IDNOs: 20, 23, and 25) and the extracellular region protein of humanFrizzled 7, Frizzled 1, and Frizzled 2 (SEQ ID NOs: 19, 22, and 25).

In view of nucleic acid expression in an eukaryotic cell andextracellular secretion of the expression product, it is preferable thata nucleotide sequence encoding a signal sequence be further included.Examples of signal sequences include a signal sequence derived from aFrizzled receptor protein, a signal sequence derived from human CD33, asignal sequence derived from human serum albumin, and a signal sequencederived from human preprotrypsin.

Examples of nucleotide sequences encoding protein precursors of theextracellular domains of mouse- and human-derived Frizzled 7, Frizzled1, and Frizzled 2 are provided below. Underlined portions indicatenucleotide sequences encoding signal sequences and non-underlinedportions indicate nucleotide sequences encoding mature sequences ofextracellular region proteins.

DNA encoding the mouse Frizzled 7 extracellularregion protein (SEQ ID NO: 32):ATGCGGGGCCCCGGCACGGCGGCGTCGCACTCGCCCCTGGGCCTCTGCGCCCTGGTGCTTGCTCTTCTGTGCGCGCTGCCCACGGACACCCGGGCTCAGCCATATCACGGCGAGAAAGGCATCTCGGTACCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGTTGTGCACGGATATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCTCTGGTAAAGGTGCAGTGTTCTCCTGAGCTACGCTTCTTCTTATGCTCTATGTACGCACCCGTGTGCACCGTGCTCGACCAAGCCATTCCTCCGTGCCGTTCCTTGTGCGAGCGCGCCCGACAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGAGCGGTTGCGCTGCGAGAACTTCCCAGTGCACGGTGCCGGCGAGATCTGCGTGGGGCAGAACACGTCCGACGGCTCCGGGGGCGCGGGCGGCAGTCCCACCGCCTACCCTACTGCTCCCT ACCTGDNA encoding the human Frizzled 7 extracellularregion protein (SEQ ID NO: 33):ATGCGGGACCCCGGCGCGGCCGCTCCGCTTTCGTCCCTGGGCCTCTGTGCCCTGGTGCTGGCGCTGCTGGGCGCACTGTCCGCGGGCGCCGGGGCGCAGCCGTACCACGGAGAGAAGGGCATCTCCGTGCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCGCTGGTGAAGGTGCAGTGTTCTCCCGAACTCCGCTTTTTCTTATGCTCCATGTATGCGCCCGTGTGCACCGTGCTCGATCAGGCCATCCCGCCGTGTCGTTCTCTGTGCGAGCGCGCCCGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCCGAGCGGCTGCGCTGCGAGAACTTCCCGGTGCACGGTGCGGGCGAGATCTGCGTGGGCCAGAACACGTCGGACGGCTCCGGGGGCCCAGGCGGCGGCCCCACTGCCTACCCTACCGCGCCCT ACCTGDNA encoding the mouse Frizzled 1 extracellularregion protein (SEQ ID NO: 34):ATGGCTGAGGAGGCGGCGCCTAGCGAGTCCCGGGCCGCCGGCCGGCTGAGCTTGGAACTTTGTGCCGAAGCACTCCCGGGCCGGCGGGAGGAGGTGGGGCACGAGGACACGGCCAGCCACCGCCGCCCCCGGGCTGATCCCCGGCGTTGGGCTAGCGGGCTGCTGCTGCTGCTTTGGTTGCTGGAGGCTCCTCTGCTTTTGGGGGTCCGAGCGCAGGCGGCGGGCCAGGTATCCGGGCCGGGCCAGCAAGCCCCGCCGCCGCCCCAGCCCCAGCAGAGCGGGCAGCAGTACAACGGCGAACGGGGCATCTCCATCCCGGACCACGGCTACTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAATCAGGAGGACGCCGGTCTGGAGGTGCACCAGTTCTACCCTCTGGTGAAGGTGCAGTGCTCCGCCGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCTGTGTGCACCGTACTGGAGCAGGCGCTACCGCCCTGCCGCTCCCTGTGCGAGCGCGCACGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACACTCAAGTGCGAGAAGTTCCCGGTGCACGGCGCAGGAGAGCTGTGCGTGGGCCAGAACACGTCCGACAAAGGCACCCCAACTCCCTCCTTGCTACCAGAGTTCTGGACCAGTAATCCGCAGCACDNA encoding the human Frizzled 1 extracellularregion protein (SEQ ID NO: 35):ATGGCTGAGGAGGAGGCGCCTAAGAAGTCCCGGGCCGCCGGCGGTGGCGCGAGCTGGGAACTTTGTGCCGGGGCGCTCTCGGCCCGGCTGGCGGAGGAGGGCAGCGGGGACGCCGGTGGCCGCCGCCGCCCGCCAGTTGACCCCCGGCGATTGGCGCGCCAGCTGCTGCTGCTGCTTTGGCTGCTGGAGGCTCCGCTGCTGCTGGGGGTCCGGGCCCAGGCGGCGGGCCAGGGGCCAGGCCAGGGGCCCGGGCCGGGGCAGCAACCGCCGCCGCCGCCTCAGCAGCAACAGAGCGGGCAGCAGTACAACGGCGAGCGGGGCATCTCCGTCCCGGACCACGGCTATTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAACCAGGAGGACGCGGGCCTGGAGGTGCACCAGTTCTACCCTCTAGTGAAAGTGCAGTGTTCCGCTGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCCGTGTGCACCGTGCTAGAGCAGGCGCTGCCGCCCTGCCGCTCCCTGTGCGAGCGCGCGCGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACGCTCAAGTGTGAGAAGTTCCCGGTGCACGGCGCCGGCGAGCTGTGCGTGGGCCAGAACACGTCCGACAAGGGCACCCCGACGCCCTCGCTGCTTCCAGAGTTCTGGACCAGCAAC CCTCAGCACDNA encoding the mouse Frizzled 2 extracellularregion protein (SEQ ID NO: 36):ATGCGGGCCCGCAGCGCCCTGCCCCGCAGCGCCCTGCCCCGCCTGCTGCTGCCACTGCTGCTGCTGCCGGCCGCCGGACCGGCCCAGTTCCACGGGGAGAAGGGCATCTCCATCCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTTGGCCACACGAACCAGGAAGACGCGGGCCTGGAGGTGCATCAGTTCTACCCGCTGGTGAAGGTGCAGTGCTCGCCCGAGCTGCGCTTCTTCCTGTGCTCCATGTACGCGCCGGTGTGCACAGTGCTGGAGCAGGCCATCCCGCCGTGCCGCTCCATCTGCGAGCGCGCGCGCCAAGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAATGGCCCGAGCGCCTCCGCTGCGAGCATTTCCCGCGTCACGGCGCGGAGCAGATCTGCGTGGGCCAGAACCACTCGGAGGACGGAGCTCCTGC GCTADNA encoding the human Frizzled 2 extracellularregion protein (SEQ ID NO: 37):ATGCGGCCCCGCAGCGCCCTGCCCCGCCTGCTGCTGCCGCTGCTGCTGCTGCCCGCCGCCGGGCCGGCCCAGTTCCACGGGGAGAAGGGCATCTCCATCCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTGGGCCACACGAACCAGGAGGACGCAGGCCTAGAGGTGCACCAGTTCTATCCGCTGGTGAAGGTGCAGTGCTCGCCCGAACTGCGCTTCTTCCTGTGCTCCATGTACGCACCCGTGTGCACCGTGCTGGAACAGGCCATCCCGCCGTGCCGCTCTATCTGTGAGCGCGCGCGCCAGGGCTGCGAAGCCCTCATGAACAAGTTCGGTTTTCAGTGGCCCGAGCGCCTGCGCTGCGAGCACTTCCCGCGCCACGGCGCCGAGCAGATCTGCGTCGGCCAGAACCACTCCGAGGACGGAGCTCCCGCGCTA

The term “nucleic acid” used in the present invention encompasses anucleic acid encoding a fusion protein of a protein comprising theextracellular cysteine-rich domain of the Frizzled receptor or a mutantthereof and the foreign protein defined above. A preferable example ofthe foreign protein is a mammalian animal-derived immunoglobulin Fcprotein, with a human Fc protein being particularly preferable. It ispreferable that such foreign protein be introduced so as to reduce orlose biological activity (ADCC and CDC in particular). An example of anucleotide sequence encoding a mutant human IgG1-derived Fc protein isshown in SEQ ID NO: 3. Further, examples of nucleotide sequencesencoding fusion proteins of the mutant human IgG1-derived Fc proteins(underlined portions) and proteins comprising the extracellularcysteine-rich domains of the mouse- or human-derived Frizzled 7, 1, and2 receptors (non-underlined portions) are shown below.

DNA encoding the fusion protein of the mouseFrizzled 7 extracellular region protein and themutant human IgG1-derived Fc protein (SEQ ID NO: 38):CAGCCATATCACGGCGAGAAAGGCATCTCGGTACCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGTTGTGCACGGATATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCTCTGGTAAAGGTGCAGTGTTCTCCTGAGCTACGCTTCTTCTTATGCTCTATGTACGCACCCGTGTGCACCGTGCTCGACCAAGCCATTCCTCCGTGCCGTTCCTTGTGCGAGCGCGCCCGACAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGAGCGGTTGCGCTGCGAGAACTTCCCAGTGCACGGTGCCGGCGAGATCTGCGTGGGGCAGAACACGTCCGACGGCTCCGGGGGCGCGGGCGGCAGTCCCACCGCCTACCCTACTGCTCCCTACCTGGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGADNA encoding the fusion protein of the humanFrizzled 7 extracellular region protein and themutant human IgG1-derived Fc protein (SEQ ID NO: 39):CAGCCGTACCACGGAGAGAAGGGCATCTCCGTGCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCGCTGGTGAAGGTGCAGTGTTCTCCCGAACTCCGCTTTTTCTTATGCTCCATGTATGCGCCCGTGTGCACCGTGCTCGATCAGGCCATCCCGCCGTGTCGTTCTCTGTGCGAGCGCGCCCGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCCGAGCGGCTGCGCTGCGAGAACTTCCCGGTGCACGGTGCGGGCGAGATCTGCGTGGGCCAGAACACGTCGGACGGCTCCGGGGGCCCAGGCGGCGGCCCCACTGCCTACCCTACCGCGCCCTACCTGGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGADNA encoding the fusion protein of the mouseFrizzled 1 extracellular region protein and themutant human IgG1-derived Fc protein (SEQ ID NO: 40):CAGGCGGCGGGCCAGGTATCCGGGCCGGGCCAGCAAGCCCCGCCGCCGCCCCAGCCCCAGCAGAGCGGGCAGCAGTACAACGGCGAACGGGGCATCTCCATCCCGGACCACGGCTACTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAATCAGGAGGACGCCGGTCTGGAGGTGCACCAGTTCTACCCTCTGGTGAAGGTGCAGTGCTCCGCCGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCTGTGTGCACCGTACTGGAGCAGGCGCTACCGCCCTGCCGCTCCCTGTGCGAGCGCGCACGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACACTCAAGTGCGAGAAGTTCCCGGTGCACGGCGCAGGAGAGCTGTGCGTGGGCCAGAACACGTCCGACAAAGGCACCCCAACTCCCTCCTTGCTACCAGAGTTCTGGACCAGTAATCCGCAGCACGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGADNA encoding the fusion protein of the humanFrizzled 1 extracellular region protein and themutant human IgG1-derived Fc protein (SEQ ID NO: 41):CAGGCGGCGGGCCAGGGGCCAGGCCAGGGGCCCGGGCCGGGGCAGCAACCGCCGCCGCCGCCTCAGCAGCAACAGAGCGGGCAGCAGTACAACGGCGAGCGGGGCATCTCCGTCCCGGACCACGGCTATTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAACCAGGAGGACGCGGGCCTGGAGGTGCACCAGTTCTACCCTCTAGTGAAAGTGCAGTGTTCCGCTGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCCGTGTGCACCGTGCTAGAGCAGGCGCTGCCGCCCTGCCGCTCCCTGTGCGAGCGCGCGCGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACGCTCAAGTGTGAGAAGTTCCCGGTGCACGGCGCCGGCGAGCTGTGCGTGGGCCAGAACACGTCCGACAAGGGCACCCCGACGCCCTCGCTGCTTCCAGAGTTCTGGACCAGCAACCCTCAGCACGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGADNA encoding the fusion protein of the mouseFrizzled 2 extracellular region protein and themutant human IgG1-derived Fc protein (SEQ ID NO: 42):CAGTTCCACGGGGAGAAGGGCATCTCCATCCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTTGGCCACACGAACCAGGAAGACGCGGGCCTGGAGGTGCATCAGTTCTACCCGCTGGTGAAGGTGCAGTGCTCGCCCGAGCTGCGCTTCTTCCTGTGCTCCATGTACGCGCCGGTGTGCACAGTGCTGGAGCAGGCCATCCCGCCGTGCCGCTCCATCTGCGAGCGCGCGCGCCAAGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAATGGCCCGAGCGCCTCCGCTGCGAGCATTTCCCGCGTCACGGCGCGGAGCAGATCTGCGTGGGCCAGAACCACTCGGAGGACGGAGCTCCTGCGCTAGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA DNA encoding the fusion protein of the humanFrizzled 2 extracellular region protein and themutant human IgG1-derived Fc protein (SEQ ID NO: 43):CAGTTCCACGGGGAGAAGGGCATCTCCATCCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTGGGCCACACGAACCAGGAGGACGCAGGCCTAGAGGTGCACCAGTTCTATCCGCTGGTGAAGGTGCAGTGCTCGCCCGAACTGCGCTTCTTCCTGTGCTCCATGTACGCACCCGTGTGCACCGTGCTGGAACAGGCCATCCCGCCGTGCCGCTCTATCTGTGAGCGCGCGCGCCAGGGCTGCGAAGCCCTCATGAACAAGTTCGGTTTTCAGTGGCCCGAGCGCCTGCGCTGCGAGCACTTCCCGCGCCACGGCGCCGAGCAGATCTGCGTCGGCCAGAACCACTCCGAGGACGGAGCTCCCGCGCTAGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Examples of nucleotide sequences encoding amino acid sequences of thecysteine rich domains (CRDs) spanning from the 1st cysteine residue onthe N-terminal side to the 10th cysteine residue of the mouse- andhuman-derived Frizzled 7, Frizzled 1, and Frizzled 2 extracellularregion proteins are shown below.

SEQ ID NO: 44: mouse Frizzled 7 CRDTGCCAGCCCATCTCCATCCCGTTGTGCACGGATATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCTCTGGTAAAGGTGCAGTGTTCTCCTGAGCTACGCTTCTTCTTATGCTCTATGTACGCACCCGTGTGCACCGTGCTCGACCAAGCCATTCCTCCGTGCCGTTCCTTGTGCGAGCGCGCCCGACAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGAGCGGTTGCGCTGCGAGAACTTCCCAGTGCACGGTGCCGGCGAGATCTGC SEQ ID NO: 45: human Frizzled 7 CRDTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCGCTGGTGAAGGTGCAGTGTTCTCCCGAACTCCGCTTTTTCTTATGCTCCATGTATGCGCCCGTGTGCACCGTGCTCGATCAGGCCATCCCGCCGTGTCGTTCTCTGTGCGAGCGCGCCCGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCCGAGCGGCTGCGCTGCGAGAACTTCCCGGTGCACGGTGCGGGCGAGATCTGC SEQ ID NO: 46: mouse Frizzled 1 CRDTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAATCAGGAGGACGCCGGTCTGGAGGTGCACCAGTTCTACCCTCTGGTGAAGGTGCAGTGCTCCGCCGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCTGTGTGCACCGTACTGGAGCAGGCGCTACCGCCCTGCCGCTCCCTGTGCGAGCGCGCACGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACACTCAAGTGCGAGAAGTTCCCGGTGCACGGCGCAGGAGAGCTGTGC SEQ ID NO: 47: human Frizzled 1 CRDTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAACCAGGAGGACGCGGGCCTGGAGGTGCACCAGTTCTACCCTCTAGTGAAAGTGCAGTGTTCCGCTGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCCGTGTGCACCGTGCTAGAGCAGGCGCTGCCGCCCTGCCGCTCCCTGTGCGAGCGCGCGCGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACGCTCAAGTGTGAGAAGTTCCCGGTGCACGGCGCCGGCGAGCTGTGC SEQ ID NO: 48: mouse Frizzled 2 CRDTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTTGGCCACACGAACCAGGAAGACGCGGGCCTGGAGGTGCATCAGTTCTACCCGCTGGTGAAGGTGCAGTGCTCGCCCGAGCTGCGCTTCTTCCTGTGCTCCATGTACGCGCCGGTGTGCACAGTGCTGGAGCAGGCCATCCCGCCGTGCCGCTCCATCTGCGAGCGCGCGCGCCAAGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAATGGCCCGAGCGCCTCCGCTGCGAGCATTTCCCGCGTCACGGCGCGGAGCAGATCTGC SEQ ID NO: 49: human Frizzled 2 CRDTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTGGGCCACACGAACCAGGAGGACGCAGGCCTAGAGGTGCACCAGTTCTATCCGCTGGTGAAGGTGCAGTGCTCGCCCGAACTGCGCTTCTTCCTGTGCTCCATGTACGCACCCGTGTGCACCGTGCTGGAACAGGCCATCCCGCCGTGCCGCTCTATCTGTGAGCGCGCGCGCCAGGGCTGCGAAGCCCTCATGAACAAGTTCGGTTTTCAGTGGCCCGAGCGCCTGCGCTGCGAGCACTTCCCGCGCCACGGCGCCGAGCAGATCTGC

Examples of the nucleotide sequences encoding the fusion proteinsdescribed above further include nucleotide sequences encoding signalsequences. Examples of signal sequences include human protein-derivedsignal sequences, such as human Frizzled 1, 2, and 7-derived signalsequences, human CD33-derived signal sequences, human serumalbumin-derived signal sequences, and human preprotrypsin-derived signalsequences.

Homologs of nucleic acids encoding the proteins can be obtained fromcDNA libraries prepared from cells or tissues that are known to expressgenes derived from mammalian animals other than humans and mice viawell-known techniques involving the use of primers or probes preparedbased on cDNAs synthesized from mRNAs encoding the human- andmouse-derived Frizzled 7, 1, and 2 genes. Examples of such techniquesinclude PCR and hybridization (e.g., Southern or Northernhybridization).

PCR stands for a polymerase chain reaction, which involves about 25 to40 cycles of a reaction cycle comprising a denaturing process fordessociating double-stranded DNA into single-stranded DNA (about 94° C.to 96° C. for about 30 seconds to 1 minute), an annealing process forbinding a primer to template single-stranded DNA (about 55° C. to 68° C.for about 30 seconds to 1 minute), and an extension process forextending a DNA strand (about 72° C. for about 30 seconds to 1 minute).Also, a pre-heating process can be carried out at about 94° C. to 95° C.for about 5 to 12 minutes prior to the denaturing process and anotherextension reaction can be carried out at 72° C. for about 7 to 15minutes after the final cycle of the extension process. PCR is carriedout using a commercially available thermal cycler in a PCR buffercontaining, for example, thermostable DNA polymerase (e.g., AmpliTaqGold® (Applied Biosystems)), MgCl₂, and dNTP (e.g., dATP, dGTP, dCTP, ordTTP) in the presence of sense and antisense primers (size: about 17 to30 bases, preferably 20 to 25 bases) and template DNA. Amplified DNA canbe separated and purified via agarose gel electrophoresis (ethidiumbromide staining).

Hybridization is a technique comprising forming a double strand with anabout 20 to 100 bases or longer label probe and detecting a targetnucleic acid. In order to enhance selectivity, hybridization can begenerally carried out under stringent conditions. Under stringentconditions, for example, hybridiztion is carried out in the presence ofabout 1 to 5×SSC at room temperature to about 40° C., and washing isthen carried out in the presence of about 0.1 to 1×SSC and 0.1% SDS atabout 45° C. to 65° C. The term “1×SSC” used herein refers to a solutioncomprising 150 mM NaCl and 15 mM Na-citrate (pH 7.0). Under suchconditions, nucleic acids having sequence identity of 80% or higher, andpreferably 85% or higher, can be detected.

The nucleic acid is inserted into a vector, and the resulting vector isused for the production of a protein as an active ingredient of thepharmaceutical composition of the present invention, or such vector isformulated into and used for a pharmaceutical composition.

Examples of vectors include plasmid, phage, and virus vectors. Examplesof plasmid vectors include, but are not limited to, E. coli-derivedplasmid (e.g., pRSET, pTZ19R, pBR322, pBR325, pUC118, and pUC119),Bacillus subtilis-derived plasmid (e.g., pUB110 and pTPS), yeast-derivedplasmid (e.g., YEp13, YEp24, and YCp50), and Ti plasmid vectors. Anexample of a phage vector is a X, phage vector. Examples of virusvectors include animal virus vectors, such as retrovirus, vacciniavirus, lentivirus, adenovirus, and adeno-associated virus vectors, andinsect virus vectors, such as a baculovirus vector.

A vector may comprise a polylinker or multicloning site to incorporatetarget DNA, and it can comprise several control elements to expresstarget DNA. Examples of control elements include promoters, enhancers,poly A addition signals, replication origins, selection markers,ribosome binding sequences, and terminators.

Examples of selection markers include drug-resistant genes (e.g.,neomycin-resistant genes, ampicillin-resistant genes,kanamycin-resistant genes, and puromycin-resistant genes) andauxotrophic complementary genes (e.g., dihydrofolate reductase (DHFR)genes, HIS3 genes, LEU2 genes, and URA3 genes).

Promoters occasionally vary depending on host cells.

Examples of host cells include, but are not limited to: bacteria of thegenus Escherichia such as E. coli, the genus Bacillus such as Bacillussubtilis, and the genus Pseudomonas such as Pseudomonas putida; yeast ofthe genus Saccharomyces such as Saccharomyces cerevisae andSchizosaccharomydces pombe, the genus Candida, and the genus Pichia;animal cells, such as CHO, COS, HEK293, NIH3T3, and NSO; insect cells,such as Sf9 and Sf21; and plant cells.

When bacterial host cells such as E. coli cells are used, examples ofpromoters include trp promoters, lac promoters, and P_(L) or P_(R)promoters.

When yeast hosts are used, examples of promoters include gall promoters,gal 10 promoters, heat shock protein promoters, MFα1 promoters, PHO5promoters, PGK promoters, GAP promoters, ADH promoters, and AOX1promoters.

When animal host cells are used, examples of promoters include SRαpromoters, SV40 promoters, LTR promoters, CMV promoters, human CMV earlygene promoters, adenovirus late promoters, vaccinia virus 7.5Kpromoters, metallothionein promoters, and polyhedral promoters.

When plant host cells are used, examples of promoters include CaMVpromoters and TMV promoters.

Examples of transformation or transfection techniques includeelectroporation, the spheroplast method, the lithium acetate method, thecalcium phosphate method, the agrobacterium method, the virus infectionmethod, the liposome method, microinjection, the gene gun method, andlipofection method.

The transformed host cells are cultured under the conditions that aresuitable for types of bacteria, yeast, animal cells, or plant cells, andtarget proteins are recovered from the cells or the culture solution.

Microorganisms are cultured with the use of a medium containing carbonsources, nitrogen sources, inorganic salts, and the like assimilable bymicroorganisms. Examples of carbon sources that can be used includecarbohydrates, such as glucose, fructose, sucrose, and starch, organicacids, such as acetic acid and propionic acid, and alcohols, such asethanol and propanol. Examples of nitrogen sources that can be usedinclude ammonium salts of inorganic acids or organic acids, such asammonia, ammonium chloride, ammonium sulfate, ammonium acetate, andammonium phosphate, peptone, meat extract, and corn steep liquor.Examples of inorganic substances that can be used include monopotassiumphosphate, dipotassium phosphate, magnesium phosphate, magnesiumsulfate, sodium chloride, ferrous sulfate, manganese sulfate, coppersulfate, and calcium carbonate.

Animal cell culture involves the use of a medium prepared by addingfetal calf serum (FCS) or the like to a basal medium such as DMEM orRPMI 1640 medium.

As described above, target proteins can be recovered via common proteinpurification techniques, such as ammonium sulfate precipitation, organicsolvent precipitation, dialysis, electrophoresis, chromatofocusing, gelfiltration chromatography, ion exchange chromatography, affinitychromatography, or HPLC.

When a vector is used for a therapeutic purpose, a vector that is notincorporated into the subject's genome and is a virus or non-virusvector capable of infecting cells but is unreplicable is preferable.Examples of such vector include an adeno-associated virus vector and anadenovirus vector. Such vector can contain a promoter, an enhancer, apolyadenylation site, a selection marker, and a reporter gene. Examplesof virus vectors include vectors described in J. Virol. 67:5911-5921,1993, Human Gene Therapy 5: 717-729, 1994, Gene Therapy 1: 51-58, 1994,Human Gene Therapy 5: 793-801, 1994, and Gene Therapy 1:165-169, 1994and modified vectors thereof. Further, examples of non-virus vectorsinclude human artificial chromosome vectors that are composed of achromosome fragment comprising human chromosome-derived centromere andtelomere. Examples of human chromosome fragments include, but are notparticularly limited to, a human chromosome 14 fragment and a humanchromosome 21 fragment (e.g., JP Patent Publication (saihyo) No.2004-031385 A and JP Patent Publication (kokai) No. 2007-295860 A). Thenucleic acid defined above is inserted into the vector and the resultingvector is administered to the bone of the subject. Alternatively, thevector is introduced into the bone tissue or cell sampled from thesubject and the resultant is then returned to the bone of the subject.Thus, the vector can be administered to the subject.

<Pharmaceutical Composition>

The present invention further provides a composition for treating a bonedisease comprising, as an active ingredient, a protein comprising theextracellular cysteine-rich domain of the Frizzled 1, Frizzled 2, orFrizzled 7 receptor or a mutant thereof, or a vector comprising anucleic acid encoding the protein.

The present invention also provides a method for treating a bone diseasecomprising administering such composition to a mammalian animal.

In the present invention, the bone disease refers to a disease thatinvolves lowering of bone mass, bone density, and/or bone strength.Examples of the bone disease include osteoporosis, osteoarthritis,articular rheumatism, malignant tumors [e.g., osteoclastoma,osteosarcoma, and multiple myeloma (wherein the following is known withrespect to multiple myeloma: Bone pain resulting from multiple myelomaoften occurs in the spinal cord and in the costa and it is occasionallyworsened by exercise. If the pain is persistent at the same region,pathologic fracture may have occurred. When a lesion exists on thespine, spinal cord compression may occur. In the case of multiplemyeloma, IL-6 is released by the multiplied tumor cells. IL-6 is alsoknown as a factor that activates osteoclasts (OAF: osteoclast activatingfactor), and osteoclasts activated by IL-6 absorb and destroy the bone.If the bone invaded with multiple myeloma is radiographed, accordingly,the bone seems to have holes (i.e., “punched-out” resorptive lesions).Also, bone destruction leads to an elevated blood calcium level, whichcauses hypercalcemia and various symptoms resulting therefrom)], bonediseases resulting from hypercalcemia, Paget's disease of bone,osteopetrosis, Camurati-engelmann's disease, arthropathy, primaryhyperthyreosis, osteopenia, osteoporosis, osteohalisteresis, rachitis,traumatic bone fracture, or fatigue bone fracture, and various bonediseases or disorders associated therewith. Osteoporosis encompassesprimary osteoporosis and secondary osteoporosis. Examples of primaryosteoporosis include postmenopausal osteoporosis and senileosteoporosis, and examples of causal diseases of secondary osteoporosisinclude endocrine diseases (e.g., hyperparathyreosis, hyperthyreosis,hypogonadism, Cushing's syndrome, somatotropin deficiency, diabetes,Addison's disease, and calcitonin deficiency), nutritional/metabolicdiseases [e.g., chronic degenerative diseases, emaciation, serious liverdiseases (primary biliary cirrhosis, in particular), gastric resection,scorbutus, malabsorption syndrome (including celiac disease),hypophosphatemia, chronic renal disease, hypercalciuria,hemochromatosis, amyloidosis, mast cell tumor, ingestion of excesssodium, insufficient calcium intake, and hypervitaminosis D and A],inflammatory diseases [e.g., articular rheumatism, periarticular bonedisease (elevated bone resorption induced by proinflammatory cytokines),and sarcoidosis], immobile diseases (e.g., systemic, bed rest,paralytic, local, or post-fracture diseases), drug-induced diseases[e.g., steroid, which is extensively used for inflammatory diseases asimmunosuppressive agents, examples of diseases treated with steroidinclude a collagen disease, asthma, inflammatory bowel diseases, andorgan transplantation. Bone loss is a serious side effect of suchtreatment techniques), methotrexate, heparin, warfarin, anticonvulsantagent, lithium, and tamoxifen], blood diseases [e.g., multiple myeloma,lymphoma, leukaemia, hemophilia, and chronic hemolytic disease],congenital diseases (e.g., dysosteogenesis, Marfan's syndrome,Kleinfelter's syndrome, congenital erythropoetic porphyria, and cysticfibrosis), and diseases resulting from other diseases [e.g., a chronicobstructive lung disease, hepatic failure, renal disease, articularrheumatism, pregnancy, hyperoxemia, and HIV infection].

According to the present invention, the term “bone disease” also refersto a bone disease caused via selective inhibition of a mineralizationprocess, and an example thereof is rachitis.

When the composition of the present invention is administered to amammalian animal with a bone disease, preferably a mammalian animal witha disease involving lowering of bone mass, bone density, and/or bonestrength, the composition specifically acts on the bone site to increasebone mass, bone density, and/or bone strength, which at least enablesincrease in the cancellous bone and thickening and proliferation of thediaphysis. Since the composition of the present invention isbone-specific, advantageously, it would develop no or substantially noside effects in other tissue.

The dosage form of the composition of the present invention (i.e., apharmaceutical preparation) is not limited, and it can be an oral orparenteral preparation. Also, the preparation may comprise othertherapeutic agents for bone diseases, in addition to the activeingredients of the present invention. Examples of such therapeuticagents include, but are not limited to, calcium preparations (e.g.,calcium L-aspartate, calcium gluconate, and calcium lactate), activevitamin D₃ preparations (e.g., alfacalcidol and calcitriol), femalehormone preparations (e.g., estriol and conjugated estrogen), calcitoninpreparations (e.g., salmon calcitonin and elcatonin), vitamin Kpreparations (e.g., menatetrenone), bisphosphonate preparations (e.g.,disodium etidronate, alendronate sodium hydrate, and sodium risedronatehydrate), selective estrogen receptor modulators (e.g, raloxifenehydrochloride), ipriflavone, and an anti-RANKL antibody.

The other therapeutic agents can be administered in combination with thecomposition of the present invention simultaneously or continuously to amammalian animal in accordance with the therapeutic regimen made by theprimary doctor. The term “continuously” used herein refers that theother therapeutic agent may be administered after the composition of thepresent invention is administered or the composition of the presentinvention may be administered after the other therapeutic agent isadministered. That is, the timings of administration for such agents areseparated. The term “simultaneously” refers that the composition of thepresent invention is administered simultaneously with the othertherapeutic agent. In such a case, the other therapeutic agent may beincorporated into the composition of the present invention to constitutea single preparation.

A preferable form is a parenteral preparation and examples thereofinclude, but are not limited to, a preparation for intravenousadministration, a preparation for intramuscular administration, apreparation for intraperitoneal administration, a preparation forsubcutaneous administration, and a preparation for local administration.Local administration includes direct administration to an injured,fractured, or damaged bone, such as the lesion, including the cranialbone, the femur, the sternum, the spondylus, and the costa. For example,the preparation may be administered in the form of a preparation fortransplantation prepared by incorporating an active ingredient into anartificial bone component, such as hydroxyapatite. Examples ofpreparations for parenteral administrations include injectionpreparations, drops, suppositories, percutaneous absorbent preparations,liposomes, and nanoparticle-encapsulated preparations.

Examples of oral preparations include tablets, pills, granules,capsules, powders, solutions, suspensions, controlled-releasepreparations, and enteric coated preparations.

When the protein of the present invention is an active ingredient, thecomposition can contain pharmaceutically acceptable excipients, carrierssuch as diluents, and additives.

Examples of carriers include physiological saline, glycerol, ethanol,almond oil, vegetable oil, sucrose, starch, and lactose.

Examples of additives include binders (e.g., pregelatinized corn starch,hydroxypropyl methylcellulose, and polyvinyl pyrrolidone), lubricants(e.g., magnesium stearate, talc, and silica), dispersants (e.g.,polyvinyl pyrrolidone and corn starch), suspensions (e.g., talc and gumArabic), emulsifiers (e.g., lecithin and gum Arabic), disintegrators(e.g., potato starch, sodium starch glycolate, and crospovidone),buffers (e.g., phosphate, acetate, citrate, and Tris salt), antioxidants(e.g., ascorbic acid and tocopherol), preservatives (e.g., sorbic acid,methyl p-hydroxybenzoate, and propyl p-hydroxybenzoate), isotonic agents(e.g., sodium chloride), and stabilizers (e.g., glycerol).

Enteric preparations can include, for example, a polymer(s) such ashydroxypropyl methylcellulose phthalate, a copolymer of methacrylicacid-methyl methacrylate, a copolymer of methacrylic acid-ethylacrylate, and hydroxypropyl acetate succinate.

The dose of the pharmaceutical preparation should be adequatelydetermined in accordance with the age, sex, body weight, and symptoms ofthe patient, the administration route, and other conditions. Forexample, it is from about 0.1 μg/kg to 100 mg/kg per day per adult, andpreferably from about 1 μg/kg to 10 mg/kg, although the dose is notlimited to such range. The pharmaceutical preparation may beadministered daily during treatment, and it may be administered atintervals of several days, two weeks, or one month.

Another active ingredient of the present invention is a vectorcomprising a nucleic acid encoding an extracellular cysteine-rich domainof the Frizzled 7, Frizzled 1, or Frizzled 2 receptor or a mutantthereof.

The vector can be administered in the same manner as in the case of atechnique or procedure employed for gene therapy. The vector may bedirectly administered to a subject (i.e., by the in vivo method).Alternatively, the vector may be introduced into a cell sampled from asubject, a transformed cell expressing the target Frizzled extracellularcysteine-rich domain may be selected, and the selected cell may then beadministered to a subject (i.e., by the ex vivo method). Examples ofgene delivery means that can be employed for administering a vector to atarget tissue or cell include a colloidal dispersion system, aliposome-induced system, and an artificial viral envelope. Examples ofdelivery means that can be employed include a macromolecule complex,nanocapsules, microspheres, beads, oil-in-water emulsions, micells,mixed micells, and liposomes. Direct vector administration can becarried out via, for example, intravenous injection (including drops),intramuscular injection, intraperitoneal injection, or subcutaneousinjection. A vector can be introduced into a cell (i.e., transformation)via a general gene introduction technique, such as the calcium phosphatemethod, the DEAE-dextran method, electroporation, or lipofection. Theamount of the vector or transformant used varies depending on theadministration route, the administration frequency, and a subject type.Such amount can be adequately determined in accordance with a techniquecommon in the art.

<Preparation of Frizzled Extracellular Cysteine-Rich Domain Knock-inMouse>

The present invention was discovered through a B-cell-specificexpression knock-in chimeric mouse used for analyzing the in vivofunctions of the extracellular cysteine-rich domain of the Frizzled 7,Frizzled 1, or Frizzled 2 receptor and a method for producing the same.

In the present invention, a knock-in chimeric mouse expressing aFrizzled 7, Frizzled 1, or Frizzled 2 extracellular cysteine-richdomain, preferably a knock-in chimeric mouse expressing a fusion proteinof a Frizzled 7, Frizzled 1, or Frizzled 2 extracellular cysteine-richdomain and Fc, can be prepared in accordance with an establishedtechnique (for example, WO 2006/78072). In order to realize moreefficient secretion and expression in mouse B cells, for example, asecretory signal sequence of Frizzled 7, Frizzled 1, or Frizzled 2 issubstituted with the secretory signal sequence of the mouse Igκ gene. Inthis case: when knocking-in of the human Frizzled 7 extracellularcysteine-rich domain or the fusion protein of human Frizzled 7extracellular cysteine-rich domain and Fc is intended, the substitutionof the region from the N-terminus to alanine-32 of the human Frizzled 7extracellular cysteine-rich domain protein (SEQ ID NO: 8) is preferable;when knocking-in of the mouse Frizzled 7 extracellular cysteine-richdomain is intended, the substitution of the region from the N-terminusto alanine-32 of the mouse Frizzled 7 extracellular cysteine-rich domainprotein (SEQ ID NO: 2) is preferable; when knocking-in of the humanFrizzled 1 extracellular cysteine-rich domain or the fusion protein ofthe human Frizzled 1 extracellular cysteine-rich domain and Fc isintended, the substitution of the region from the N-terminus toalanine-72 of the human Frizzled 1 extracellular cysteine-rich domainprotein (SEQ ID NO: 16) is preferable; when knocking-in of the mouseFrizzled 1 extracellular cysteine-rich domain is intended, thesubstitution of the region from the N-terminus to alanine-71 of themouse Frizzled 1 extracellular cysteine-rich domain protein (SEQ ID NO:12) is preferable; and when knocking-in of the human or mouse Frizzled 2extracellular cysteine-rich domain or the fusion protein of the human ormouse Frizzled 2 extracellular cysteine-rich domain and Fc is intended,the substitution of the region from the N-terminus to alanine-28 of themouse Frizzled 2 extracellular cysteine-rich domain protein (SEQ ID NO:59) is preferable. Since the amino acid sequences of the cysteine richdomains of human and mouse Frizzled 2 are identical to each other,either a human- or mouse-derived amino acid sequence may be used. When afusion protein with Fc is to be expressed, use of an Fc mutant (hFcm)prepared by varying part of human IgG1-derived Fc into an ADCC and CDCactivity-lowered form is preferable.

A knock-in chimeric mouse expressing a human or mouse Frizzled 7,Frizzled 1, or Frizzled 2 extracellular cysteine-rich domain or a fusionprotein of a human or mouse Frizzled 7, Frizzled 1, or Frizzled 2extracellular cysteine-rich domain and hFcm, and a control chimericmouse prepared with the use of ES cells into which a foreign cDNAexpression unit has not been inserted or only the hFcm expression unithas been exclusively inserted can be subjected to, for example,pathologic analysis of tissue, immunohistochemical analysis, biochemicalexamination of serum samples, or assay of blood cell components toidentify changes resulting from the expression of the Frizzled 7,Frizzled 1, or Frizzled 2 extracellular cysteine-rich domain. InExamples 2 and 13 below, whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, whitening and hardeningof the spondylus, and hardening of the costa were more significantlyobserved in Example 2 (2-1) as phenotypes specific for a knock-inchimeric mouse expressing a mouse Frizzled 7 extracellular cysteine-richdomain compared with the control chimeric mouse. The increased femoralcancellous bone and the increased sternal cancellous bone were observedvia observation of hematoxylin-eosin (H&E)-stained pathological sectionsin Example 2 (2-2). The increased tibial bone density was observed viaX-ray photography of the tibia in Example 2 (2-3). The increased tibialbone volume/tissue volume was observed in Example 13 (13-2-2). Theincreased mineral apposition rate, the increased mineralization surface,and the increased bone formation rate of the tibia were observed inExample 13 (13-2-4). The increased maximum load of femur was observed inExample 13 (13-3). The increased bone volume/tissue volume, theincreased trabecular thickness, the increased trabecular number, thedecreased trabecular separation, and the decreased trabecular spacing inthe cancellous bone region of the proximal tibial metaphysis wereobserved in Example 13 (13-4). In Example 14 below, whitening of thefemur, whitening of the sternum, whitening and hardening of the cranium,and hardening of the costa were more significantly observed in Example14 (14-2) and (14-5) as phenotypes specific for a knock-in chimericmouse expressing a human Frizzled 7 extracellular cysteine-rich domaincompared with the control chimeric mouse. The increased maximum load offemur was observed in Example 14 (14-3). The increased bonevolume/tissue volume, the increased trabecular thickness, the increasedtrabecular number, the decreased trabecular separation, and thedecreased trabecular spacing in the cancellous bone region of the distalfemoral metaphysis were observed in Example 14 (14-4). In Example 14(14-6), the increased femoral cancellous bone, the thickened femoraldiaphyseal wall, and the increased sternal cancellous bone were observedvia observation of H&E stained pathological sections. In Examples 5 and16 below, whitening of the femur, whitening of the sternum, whiteningand hardening of the cranium, hardening of the spondylus, and hardeningof the costa were observed in Example 5 (5-1) as phenotypes specific fora knock-in chimeric mouse expressing a Frizzled 1 extracellularcysteine-rich domain. The increased tibial bone density was observed viaX-ray photography of the tibia in Example 5 (5-2). The thickened femoraldiaphyseal wall, the increased femoral cancellous bone, and theincreased sternal cancellous bone were observed via observation of H&Estained pathological sections in Example 5 (5-4). The increased tibialbone volume/tissue volume was observed in Example 16 (16-2-2). Theincreases in a mineral apposition rate, a mineralization surface, and abone formation rate of the tibia were observed in Example 16 (16-2-4).The increased maximum load of femur was observed in Example 16 (16-3).The increased bone volume/tissue volume, the increased trabecularthickness, the increased trabecular number, the decreased trabecularseparation, and the decreased trabecular spacing in the cancellous boneregion of the distal femoral metaphysis were observed in Example 16(16-4). In Examples 10 and 19 below, whitening of the femur, whiteningof the sternum, whitening and hardening of the cranium, hardening of thespondylus, and hardening of the costa were observed in Example 10 (10-1)as phenotypes specific for a knock-in chimeric mouse expressing aFrizzled 2 extracellular cysteine-rich domain. The thickened femoraldiaphyseal wall was observed via observation of H&E stained pathologicalsections in Example 10 (10-2). The increased tibial bone volume/tissuevolume was observed in Example 19 (19-4-2). The increased mineralapposition rate, the increased mineralization surface, and the increasedbone formation rate of the tibia were observed in Example 19 (19-4-4).The decreased osteoclast number and the decreased osteoclast surfacewere observed in Example 19-4-5. The increased maximum load of femur wasobserved in Example 19 (19-5). The increased bone volume/tissue volume,the increased trabecular thickness, the increased trabecular number, thedecreased trabecular separation, and the decreased trabecular spacing inthe cancellous bone region of the distal femoral metaphysis wereobserved in Example 19 (19-6).

A knock-in chimeric mouse expressing a Frizzled 7, Frizzled 1, orFrizzled 2 extracellular cysteine-rich domain or a fusion protein of theFrizzled 7, Frizzled 1, or Frizzled 2 extracellular cysteine-rich domainand hFcm can be prepared in accordance with an established method (forexample, WO 2006/78072). Whether or not the nucleic acid inserted in theknock-in ES cell-derived cells (i.e., the Frizzled 7, Frizzled 1, orFrizzled 2 extracellular cysteine-rich domain or a fusion protein of theFrizzled 7, Frizzled 1, or Frizzled 2 extracellular cysteine-rich domainand hFcm) is expressed can be detected via, for example, RT-PCRinvolving the use of RNA derived from the cell of interest, Northernblotting, enzyme-linked immunosorbent assay (ELISA) using an antibodyagainst the Frizzled 7, Frizzled 1, or Frizzled 2 extracellularcysteine-rich domain or hFcm, or Western blotting.

Hereafter, the present invention is described in greater detail withreference to the examples, although the technical scope of the presentinvention is not limited thereto. Frizzled 7, Frizzled 1, or Frizzled 2is denoted by FZD7, FZD1, or FZD2, respectively.

EXAMPLES Example 1 Preparation of USmFZD7crd-hFcm KI Chimeric Mouse

In accordance with the method described in the examples of WO2006/78072, a pUSmFZD7crd-hFcm KI vector was prepared from mouseFZD7-cDNA (a 1,719-bp sequence comprising a region from an initiationcodon to a termination codon, SEQ ID NO: 1) and human IgG1 Fcmutant-cDNA (a 702-bp sequence comprising a region from a linkersequence to a termination codon inserted to bind to the FZD7extracellular cysteine-rich domain, SEQ ID NO: 3).

The mouse FZD7 signal sequence, a CRD (the cystein-rich-domain), and aregion located downstream of a CRD comprising the 7-transmembrane domainin SEQ ID NO: 1 are marked by a single underline, a solid box, and adouble underline, respectively, based on the information regarding theGenBank Accession Numbers: NM_008057.2 and NP_032083.2.

SEQ ID NO: 1:ATGCGGGGCCCCGGCACGGCGGCGTCGCACTCGCCCCTGGGCCTCTGCGCCCTGGTGCTTGCTCTTCTGTGCGCGCTGCCCACGGACACCCGGGCT

CCAGACCCACCTTTCACTGCGATGTCCCCCTCAGATGGCAGAGGCCGCTTGTCTTTCCCCTTCTCGTGTCCGCGCCAGCTCAAGGTGCCCCCCTACCTGGGCTACCGCTTCCTAGGTGAGCGTGACTGCGGTGCCCCGTGTGAGCCGGGCCGTGCTAACGGCCTCATGTACTTTAAAGAAGAGGAGAGACGGTTCGCCCGCCTCTGGGTGGGTGTGTGGTCAGTGCTGTGCTGCGCCTCGACGCTCTTCACGGTGCTCACCTACCTAGTGGACATGCGTCGCTTCAGCTATCCAGAGCGACCCATCATCTTCCTGTCGGGTTGCTACTTCATGGTGGCAGTGGCGCACGTGGCAGGCTTCCTGCTAGAGGACCGTGCCGTGTGCGTGGAGCGCTTCTCGGACGATGGCTACCGCACGGTGGCGCAGGGCACCAAGAAGGAGGGCTGCACCATCCTCTTCATGGTGCTTTACTTCTTCGGTATGGCCAGCTCCATCTGGTGGGTCATTCTGTCCCTCACTTGGTTCCTGGCAGCTGGCATGAAGTGGGGCCACGAGGCCATCGAGGCCAACTCGCAGTACTTTCATCTGGCCGCGTGGGCTGTGCCAGCGGTCAAGACAATCACCATTTTGGCCATGGGCCAGGTGGATGGTGACCTACTCAGTGGAGTGTGCTACGTGGGCCTGTCTAGTGTGGATGCATTGCGGGGCTTCGTGCTGGCGCCCTTGTTCGTCTACCTCTTCATCGGGACGTCCTTCCTGTTGGCCGGCTTTGTGTCTCTCTTTCGCATCCGCACCATCATGAAGCACGACGGCACCAAGACAGAGAAGCTGGAGAAGCTGATGGTGCGCATCGGCGTCTTCAGCGTGCTCTACACGGTGCCGGCCACCATCGTGTTGGCCTGCTACTTTTATGAGCAGGCCTTCCGAGAGCACTGGGAACGCACCTGGCTCCTGCAGACTTGCAAGAGCTACGCTGTGCCCTGCCCTCCGGGCCACTTCTCTCCCATGAGCCCCGACTTTACAGTCTTCATGATCAAGTACCTGATGACCATGATCGTGGGCATCACTACGGGCTTCTGGATCTGGTCGGGCAAGACCCTGCAGTCATGGCGTCGCTTCTACCACAGACTCAGCCACAGCAGCAAGGGGGAAACTGCGGTATGA

The amino acid sequence encoded by SEQ ID NO: 1 (572 amino acids, SEQ IDNO: 2) is shown below.

SEQ ID NO: 2: MRGPGTAASHSPLGLCALVLALLCALPTDTRA

PDPPFTAMSPSDGRGRLSFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEERRFARLWVGVWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYFMVAVAHVAGFLLEDRAVCVERFSDDGYRTVAQGTKKEGCTILFMVLYFFGMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAVKTITILAMGQVDGDLLSGVCYVGLSSVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVLACYFYEQAFREHWERTWLLQTCKSYAVPCPPGHFSPMSPDFTVFMIKYLMTMIVGITTGFWIWSGKTLQSWRRFYHRLSHSSKGETAV

SEQ ID NOs: 3 and 4 show the cDNA sequence and the amino acid sequenceof the human IgG1-derived Fc mutant (hFcm). A cDNA region and the aminoacid sequence region (amino acids from the N-terminus before and aftermutation, L→A, G→A) with lowered ADCC activity mutated based on knowninformation (Tawara, T., et al., J. Immunology, 180, 2294-2298, 2008;Gross, J. A., et al., Immunity, 15, 289-302, 2001; and WO 02/094852) inthe original human IgG1-derived Fc region are marked by a doubleunderline, a cDNA region and an amino acid sequence region (amino acidsequences before and after mutation, K→A, P→S) mutated to have adecreased CDC activity are marked by a solid box, and a linker sequence(including the SfoI recognition sequence) added to the 5′ terminus ofthe original human IgG1-derived Fc sequence so as to bind to the Cterminal amino acid in the FZD7 extracellular cysteine-rich domain ismarked by a single underline. In addition to the above method, the 116thresidue from the N-terminus of the sequence as shown in SEQ ID NO: 4 canbe mutated from A to S in order to decrease the CDC activity, based onknown information (Gross, J. A., et al., Immunity, 15, 289-302, 2001).

SEQ ID NO: 3:GCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA

CCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The amino acid sequence encoded by SEQ ID NO: 3 (233 amino acids, SEQ IDNO: 4) is shown below.

SEQ ID NO: 4:AEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

A polynucleotide sequence comprising a region from the initiation codonto the termination codon of the pUSmFZD7crd-hFcm KI vector expressionunit (SEQ ID NO: 5; a 1,462-bp sequence comprising a mouse Igκ signalsequence containing an intron region substituted with the mouse FZD7signal sequence (a region marked by a single underline) and a mouseFZD7crd-hFcm sequence downstream thereof. A region marked by a solid boxrepresents a mouse Frizzled 7 extracellular cysteine-rich domain, aregion marked by a double underline represents hFcm, and the amino acidsequence encoded by the cDNA (SEQ ID NO: 6; a sequence composed of 406amino acids; a region marked by a single underline represents a mouseIgκ signal sequence, a region marked by a solid box represents a mouseFrizzled 7 extracellular cysteine-rich domain, and a region marked by adouble underline represents hFcm) are shown below. As informationregarding the mouse Igκ signal sequence containing an intron region, thegenomic sequence located upstream of MUSIGKVR1 obtained from the GenBank(Accession Number: K02159) was obtained from the UCSC mouse genomedatabase.

SEQ ID NO: 5:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTTATCATTCCAGGTTCCACTGGC

GCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 6: METDTLLLWVLLLWVPGSTG

AEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

With the use of the pUSmFZD7crd-hFcm KI vector, the USmFZD7crd-hFcm KIchimeric mice expressing a fusion protein of the mouse Frizzled 7extracellular cysteine-rich domain and human Fcm in a B-cell-specificmanner were prepared in accordance with the method described in theexamples of WO 2006/78072.

Control chimeric mice used in Examples 2, 13, 14, 16, 17, and 19 belowwere prepared in accordance with the method described in the examples ofWO 2006/78072.

Example 2 Analysis of USmFZD7crd-hFcm KI Chimeric Mouse 2-1. NecropsyFinding

The chimeric mice prepared in Example 1 were subjected to necropsy atage of 16 weeks, and the spleen, the liver, the kidney, the adrenalgland, the stomach, the small intestine, the appendix, the largeintestine, the pancreas, the mesenteric lymph node, the female/malereproductive organ, the thymic gland, the lung, the heart, the brain,the muscle, the skin, the femur, the sternum, the cranium, thespondylus, and the costa were observed. As a result, whitening of thefemur, whitening of the sternum, whitening and hardening of the cranium,whitening and hardening of the spondylus, and hardening of the costawere more significantly observed as characteristic changes in theUSmFZD7crd-hFcm KI chimeric mice compared with the control mice. Inaddition, spleen enlargement was observed in approximately a half of theUSmFZD7crd-hFcm KI chimeric mice. The number of mice exhibiting changesis described below.

2-1-1. Femur

Whitening was observed more significant in all the 20 USmFZD7crd-hFcm KIchimeric mice subjected to necropsy compared with 29 control mice.

2-1-2. Sternum

Whitening was observed more significant in all the 20 USmFZD7crd-hFcm KIchimeric mice subjected to necropsy compared with 29 control mice.

2-1-3. Cranium

Whitening was observed more significant in 16 mice and hardening wasobserved more significant in 18 mice among all the 20 USmFZD7crd-hFcm KIchimeric mice subjected to necropsy compared with 29 control mice.

2-1-4. Spondylus

Whitening was observed more significant in a mouse and hardening wasobserved more significant in 10 mice among all the 20 USmFZD7crd-hFcm KIchimeric mice subjected to necropsy compared with 29 control mice.

2-1-5. Costa

Hardening was observed more significant in 7 mice among all the 20USmFZD7crd-hFcm KI chimeric mice subjected to necropsy compared with 29control mice.

2-1-6. Spleen

Enlargement was observed more significant in 11 mice among all the 20USmFZD7crd-hFcm KI chimeric mice subjected to necropsy compared withcontrol mice. Similar changes were observed in 4 of 29 control mice.

Thus, whitening of the femur, whitening of the sternum, whitening andhardening of the cranium, whitening and hardening of the spondylus, andhardening of the costa may have been induced by overexpression of themouse FZD7 extracellular cysteine-rich domain-human Fc mutant fusionconstructs.

2-2. Pathological Finding

H&E stained pathological sections of the liver, the kidney, the heart,the lung, the spleen, the thymic gland, the mesenteric lymph node, thepancreas, the brain, the adrenal gland, the spermary (in the case ofmale mice), the ovary (in the case of female mice), the femur, thesternum, the stomach, the duodenum, the jejunum, the ileum, theappendix, the colon, the spinal cord, the aorta, the skeletal muscle,and the skin obtained from seven 16-week-old control chimeric mice andtwenty USmFZD7crd-hFcm KI chimeric mice were observed. As a result,increased femoral cancellous bone and the increased sternal cancellousbone were observed in all the USmFZD7crd-hFcm KI chimeric mice (FIG. 1and FIG. 2). Only one control mouse was observed to exhibit similarchanges as described above. There were no significant changes in organsand tissues other than bones compared with control mice.

The above results demonstrate that the increased femoral cancellous boneand the increased sternal cancellous bone may have been induced byoverexpression of the mouse FZD7 extracellular cysteine-richdomain-human Fc mutant fusion constructs.

2-3. Analysis of X-Ray Photographs of Tibia

X-ray photographs (μFX-1000, FUJIFILM) of the tibiae obtained from16-week-old control chimeric mice (5 female mice and 4 male mice) andthe USmFZD7crd-hFcm KI chimeric mice (7 female mice and 6 male mice)were prepared (FIG. 3 and FIG. 4).

In the obtained X-ray photographs of the tibiae, whitening was moreadvanced in both female and male USmFZD7crd-hFcm KI chimeric micecompared with the control mice.

The above results demonstrate that whitening of the tibia may have beeninduced by overexpression of the mouse FZD7 extracellular cysteine-richdomain-human Fc mutant fusion constructs.

2-4. Blood Cell Analysis

Fourteen 8-week-old USmFZD7crd-hFcm KI female chimeric mice and six8-week-old USmFZD7crd-hFcm KI male chimeric mice, fifteen 8-week-oldfemale control mice and nine 8-week-old male control mice, fourteen15-week-old USmFZD7crd-hFcm KI female chimeric mice and six 15-week-oldUSmFZD7crd-hFcm KI male chimeric mice, and fourteen 15-week-old femalecontrol mice and eleven 15-week-old male control mice were subjected toorbital blood sampling using a glass capillary under ether anesthesia,and the obtained blood samples were subjected to blood componentanalysis using ADVIA120 (Bayer Medical Ltd.) (blood components:erythrocyte counts, hemoglobin, hematocrit, MCH, MCHC, reticulocytecounts, leukocyte counts, blood platelet counts, lymphocyte counts,neutrophil counts, monocyte counts, eosinophil counts, and basophilcounts). As a result, values obtained with the use of theUSmFZD7crd-hFcm KI chimeric mice did not show significant changescompared with the control mice at ages of 8 weeks and 15 weeks.

2-5. Biochemical Analysis of Serum

Fourteen USmFZD7crd-hFcm KI female chimeric mice, 6 USmFZD7crd-hFcm KImale chimeric mice, sixteen female control mice, and fourteen malecontrol mice were exsanguinated under ether anesthesia at age of 16weeks to prepare serum samples. With the use of Hitachi 7180 (HitachiScience Systems Ltd.), serum samples were subjected to biochemicalanalysis (LDH activity, GOT activity, GPT activity, CK activity, ALPactivity, AMY activity, LAP activity, LIP activity, T-CHO concentration,F-CHO concentration, LDL-CHO concentration, HDL-CHO concentration, TGconcentration, PL concentration, GLU concentration, GA %, UAconcentration, BUN concentration, CREA concentration, T-BILconcentration, D-BIL concentration, TP concentration, ALB concentration,A/G ratio, IP concentration, Ca concentration, Mg concentration, Naconcentration, K concentration, Cl concentration, Fe concentration, UIBCconcentration, and TIBC concentration). As a result, the values obtainedwith the use of the USmFZD7crd-hFcm KI chimeric mice were notsignificantly different from those of the control mice.

2-6. Confirmation of Expression of the Fusion Protein of Mouse FZD7Extracellular Cysteine-Rich Domain and Human Fc Mutant inUSmFZD7crd-hFcm KI Chimeric Mice

2-6-1. ELISA Assay of the Fusion Protein of Mouse FZD7 ExtracellularCysteine-Rich Domain and Human Fc Mutant Using Serum Obtained fromUSmFZD7crd-hFcm KI Chimeric Mice

The fusion protein of the mouse FZD7 extracellular cysteine-rich domainand the human Fc mutant existing in the blood sera of 16-week-oldUSmFZD7crd-hFcm KI chimeric mice (14 female mice and 6 male mice) wasdetected via ELISA.

In order to assay the concentration of the fusion protein of the FZD7extracellular cysteine-rich domain and the human Fc mutant in the serumvia ELISA, a test sample or a control sample (Recombinant MouseFrizzled-7/Fc Chimera, R & D Systems, Product Number: 198-FZ) wasapplied to a 96-well plate (Maxi Soap, Corning) on which anti-Human IgG(γ-Chain Specific, SIGMA, Product Number: 13382) has been immobilized,incubation was carried out at room temperature for 30 minutes, the platewas washed three times with T-PBS(−), peroxidase-labelled antibodies(anti-Human IgG (Fc fragment) peroxidase conjugates developed in goat,Product Number: A0170, SIGMA) were added, and incubation was thencarried out at room temperature for 30 minutes. Thereafter, the platewas washed four times with T-PBS(−), a color was developed using aSumilon peroxidase color-developing kit (Product Number: ML-1120T,Sumitomo Bakelite Co. Ltd.), and the absorbance at 450 nm was assayed todetermine the concentration in the serum.

As a result, the average concentration among 14 female mice was 201.7μg/ml, that among 6 male mice was 168.4 and the concentrations assayedwith the use of the serum samples obtained from 5 female control miceand a male control mouse were lower than the detection limit.

The above results suggest that the fusion protein of the mouse FZD7extracellular cysteine-rich domain and the human Fc mutant is expressedin vivo and circulated in the blood.

2-6-2. Western Analysis Using Serum Obtained from USmFZD7crd-hFcm KIChimeric Mice

Serum samples obtained from 16-week-old USmFZD7crd-hFcm KI chimeric miceand control chimeric mice were subjected to Western analysis using humanIgG-recognizing rabbit polyclonal antibodies. As samples used forWestern analysis, 50 μl of serum samples were applied to Protein Gcolumns (resin volume: about 100 μl, GE Healthcare) in advance,nonspecific adsorbates were removed, and 1.25 μl of serum samples and2.5 μl equivalent of resin were used as samples to be analyzed. As aresult, a main band specific for the USmFZD7crd-hFcm KI chimeric micewas detected at around 60 kDa under reducing conditions (FIG. 5). Themolecular weight determined via this analysis was larger than thatdeduced based only on the amino acid sequence (42.8 kDa under reducingconditions); however, the fusion protein of interest comprised 3 each ofN-linked and O-linked glycosylation prediction sites, which indicates anincreased molecular weight via glycosylation.

The above results suggest that the USmFZD7crd-hFcm KI chimeric miceprepared in this experiment express a fusion protein of the mouse FZD7extracellular cysteine-rich domain and the human Fc mutant.

Example 3 Preparation of UShFZD7crd-hFcm KI Chimeric Mouse

A pUShFZD7crd-hFcm KI vector was prepared from human FZD7-cDNA (SEQ IDNO: 7) and human IgG1 Fc mutant-cDNA (SEQ ID NO: 3) in accordance withthe method described in Example 1.

The human FZD7 signal sequence, CRD, and a region located downstream ofa CRD comprising the 7-transmembrane domain in SEQ ID NO: 7 are markedby a single underline, a solid box, and a double underline,respectively, based on the information regarding the GenBank AccessionNumbers: NM_003507.1 and NP_003498.1.

SEQ ID NO: 7:ATGCGGGACCCCGGCGCGGCCGCTCCGCTTTCGTCCCTGGGCCTCTGTGCCCTGGTGCTGGCGCTGCTGGGCGCACTGTCCGCGGGCGCCGGGGCG

CCGGACCTGCCCTTCACCGCGCTGCCCCCGGGGGCCTCAGATGGCAGGGGGCGTCCCGCCTTCCCCTTCTCATGCCCCCGTCAGCTCAAGGTGCCCCCGTACCTGGGCTACCGCTTCCTGGGTGAGCGCGATTGTGGCGCCCCGTGCGAACCGGGCCGTGCCAACGGCCTGATGTACTTTAAGGAGGAGGAGAGGCGCTTCGCCCGCCTCTGGGTGGGCGTGTGGTCCGTGCTGTGCTGCGCCTCGACGCTCTTTACCGTTCTCACCTACCTGGTGGACATGCGGCGCTTCAGCTACCCAGAGCGGCCCATCATCTTCCTGTCGGGCTGCTACTTCATGGTGGCCGTGGCGCACGTGGCCGGCTTCCTTCTAGAGGACCGCGCCGTGTGCGTGGAGCGCTTCTCGGACGATGGCTACCGCACGGTGGCGCAGGGCACCAAGAAGGAGGGCTGCACCATCCTCTTCATGGTGCTCTACTTCTTCGGCATGGCCAGCTCCATCTGGTGGGTCATTCTGTCTCTCACTTGGTTCCTGGCGGCCGGCATGAAGTGGGGCCACGAGGCCATCGAGGCCAACTCGCAGTACTTCCACCTGGCCGCGTGGGCCGTGCCCGCCGTCAAGACCATCACTATCCTGGCCATGGGCCAGGTAGACGGGGACCTGCTGAGCGGGGTGTGCTACGTTGGCCTCTCCAGTGTGGACGCGCTGCGGGGCTTCGTGCTGGCGCCTCTGTTCGTCTACCTCTTCATAGGCACGTCCTTCTTGCTGGCCGGCTTCGTGTCCCTCTTCCGTATCCGCACCATCATGAAACACGACGGCACCAAGACCGAGAAGCTGGAGAAGCTCATGGTGCGCATCGGCGTCTTCAGCGTGCTCTACACAGTGCCCGCCACCATCGTCCTGGCCTGCTACTTCTACGAGCAGGCCTTCCGCGAGCACTGGGAGCGCACCTGGCTCCTGCAGACGTGCAAGAGCTATGCCGTGCCCTGCCCGCCCGGCCACTTCCCGCCCATGAGCCCCGACTTCACCGTCTTCATGATCAAGTACCTGATGACCATGATCGTCGGCATCACCACTGGCTTCTGGATCTGGTCGGGCAAGACCCTGCAGTCGTGGCGCCGCTTCTACCACAGACTTAGCCACAGCAGCAAGGGGGAGACTGCGGTATGA

The amino acid sequence encoded by SEQ ID NO: 7 (574 amino acids, SEQ IDNO: 8) is shown below.

SEQ ID NO: 8: MRDPGAAAPLSSLGLCALVLALLGALSAGAGA

PDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEERRFARLWVGVWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYFMVAVAHVAGFLLEDRAVCVERFSDDGYRTVAQGTKKEGCTILFMVLYFFGMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAVKTITILAMGQVDGDLLSGVCYVGLSSVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVLACYFYEQAFREHWERTWLLQTCKSYAVPCPPGHFPPMSPDFTVFMIKYLMTMIVGITTGFWIWSGKTLQSWRRFYHRLSHSSKGETAV

A polynucleotide sequence comprising a region from the initiation codonto the termination codon of the pUShFZD7crd-hFcm KI vector expressionunit (SEQ ID NO: 9; a 1,462-bp sequence comprising a mouse Igκ signalsequence containing an intron region (a region marked by a singleunderline) substituted with the human FZD7 signal sequence and the humanFZD7crd-hFcm sequence located downstream thereof; wherein the regionmarked by a solid box represents the human Frizzled 7 extracellularcysteine-rich domain and the region marked by a double underlinerepresents hFcm) and the amino acid sequence encoded by the cDNA (SEQ IDNO: 10; a sequence comprising 406 amino acids; wherein the region markedby a single underline represents the mouse Igκ signal sequence, theregion marked by a solid box represents the human Frizzled 7extracellular cysteine-rich domain, and the region marked by a doubleunderline represents hFcm) are shown below. Information regarding themouse Igκ signal sequence containing an intron region was obtained fromthe UCSC mouse genome database as the genome sequence located upstreamof MUSIGKVR1 obtained from the GenBank (Accession Number: K02159).

SEQ ID NO: 9:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTTATCATTCCAGGTTCCACTGGC

GCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGGFCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 10: METDTLLLWVLLLWVPGSTG

AEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The UShFZD7crd-hFcm KI chimeric mice expressing a fusion protein of thehuman Frizzled 7 extracellular cysteine-rich domain and human Fcm in aB-cell-specific manner are prepared with the use of the pUShFZD7crd-hFcmKI vector in accordance with the method described in the examples of WO2006/78072.

Control chimeric mice into which no foreign cDNA expression unit hasbeen inserted are prepared in accordance with the method described inthe examples of WO 2006/78072.

Example 4 Preparation of USmFZD1crd-hFcm KI Chimeric Mouse

A pUSmFZD1crd-hFcm KI vector was prepared from mouse FZD1-cDNA (a1,929-bp sequence comprising a region from an initiation codon to atermination codon; SEQ ID NO: 11) and human IgG1 Fc mutant-cDNA (SEQ IDNO: 3) in accordance with the method described in Example 1.

The mouse FZD1 signal sequence, a CRD (the cystein-rich-domain), and aregion located downstream of a CRD comprising the 7-transmembrane domainin SEQ ID NO: 11 are marked by a single underline, a solid box, and adouble underline, respectively, based on the information regarding theGenBank Accession Numbers: NM_021457.2 and NP_067432.2.

SEQ ID NO: 11:ATGGCTGAGGAGGCGGCGCCTAGCGAGTCCCGGGCCGCCGGCCGGCTGAGCTTGGAACTTTGTGCCGAAGCACTCCCGGGCCGGCGGGAGGAGGTGGGGCACGAGGACACGGCCAGCCACCGCCGCCCCCGGGCTGATCCCCGGCGTTGGGCTAGCGGGCTGCTGCTGCTGCTTTGGTTGCTGGAGGCTCCTCTGCTTTTGGGGGTCCGAGCG

GGCGGCGGTGGTTACCGCGGCGGCTACCCGGGGGGTGCCGGGACGGTGGAGCGGGGAAAGTTCTCCTGCCCGCGCGCCCTCAGGGTGCCCTCCTACCTCAACTACCACTTTCTGGGGGAGAAGGACTGCGGCGCACCCTGCGAACCCACCAAGGTTTACGGGCTCATGTACTTCGGGCCAGAGGAGCTGCGCTTCTCGCGCACCTGGATAGGCATCTGGTCCGTGCTGTGCTGCGCCTCCACGCTCTTCACGGTGCTCACGTACCTAGTGGACATGCGGCGCTTCAGCTACCCGGAACGGCCCATCATTTTCCTGTCCGGCTGTTACACAGCGGTGGCGGTGGCCTACATCGCTGGCTTTCTGTTGGAGGACCGGGTGGTGTGCAACGACAAGTTTGCAGAGGACGGGGCGCGCACGGTGGCGCAGGGCACTAAGAAAGAAGGCTGCACTATACTCTTTATGATGCTCTACTTCTTCAGCATGGCCAGCTCCATCTGGTGGGTGATCCTGTCCCTCACCTGGTTCCTGGCAGCCGGCATGAAGTGGGGCCACGAAGCCATCGAGGCCAACTCACAGTATTTCCATTTAGCCGCCTGGGCTGTGCCAGCCATCAAAACTATAACCATCTTGGCGTTGGGCCAGGTGGATGGCGACGTACTGAGCGGAGTGTGTTTTGTGGGGCTCAACAACGTGGACGCACTGCGTGGCTTTGTGCTGGCGCCTCTCTTCGTTTATCTGTTCATTGGCACTTCTTTCCTGCTGGCCGGTTTCGTGTCACTCTTCCGCATCCGCACCATCATGAAGCATGACGGCACCAAGACAGAGAAGCTGGAGAAGCTCATGGTGCGCATCGGAGTCTTCAGTGTCCTCTACACTGTGCCGGCCACCATCGTCATCGCCTGCTACTTCTATGAACAGGCCTTTCGGGACCAGTGGGAGCGCAGCTGGGTGGCCCAGAGCTGCAAGAGTTATGCCATCCCTTGCCCTCACCTCCAGGGAGGTGGAGGAGTCCCACCACACCCGCCCATGAGCCCAGACTTTACAGTCTTCATGATCAAGTATCTCATGACGCTGATTGTGGGCATCACATCGGGCTTCTGGATCTGGTCCGGCAAGACACTGAATTCCTGGAGGAAGTTCTACACGAGGCTTACCAACAGCAAACAGGGGGAGACTACCGTCTGA

The amino acid sequence encoded by SEQ ID NO: 11 (642 amino acids, SEQID NO: 12) is shown below.

SEQ ID NO: 12:MAEEAAPSESRAAGRLSLELCAEALPGRREEVGHEDTASHRRPRADPRRWASGLLLLLWLLEAPLLLGVRA

GGGGYRGGYPGGAGTVERGKFSCPRALRVPSYLNYHFLGEKDCGAPCEPTKVYGLMYFGPEELRFSRTWIGIWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYTAVAVAYIAGFLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVGLNNVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVIACYFYEQAFRDQWERSWVAQSCKSYAIPCPHLQGGGGVPPHPPMSPDFTVFMIKYLMTLIVGITSGFWIWSGKTLNSWRKFYTRLTNSKQGETTV

A polynucleotide sequence comprising a region from the initiation codonto the termination codon of the pUSmFZD1crd-hFcm KI vector expressionunit (SEQ ID NO: 13; a 1,534-bp sequence comprising a mouse Igκ signalsequence containing an intron region (a region marked by a singleunderline) substituted with the mouse FZD1 signal sequence and the mouseFZD1crd-hFcm sequence located downstream thereof; wherein the regionmarked by a solid box represents the mouse Frizzled 1 extracellularcysteine-rich domain and the region marked by a double underlinerepresents hFcm) and the amino acid sequence encoded by the cDNA (SEQ IDNO: 14; 430 amino acids; wherein the region marked by a single underlinerepresents the mouse Igκ signal sequence, the region marked by a solidbox represents the mouse Frizzled 1 extracellular cysteine-rich domain,and the region marked by a double underline represents hFcm) are shownbelow. Information regarding the mouse Igκ signal sequence containing anintron region was obtained from the UCSC mouse genome database as thegenome sequence located upstream of MUSIGKVR1 obtained from the GenBank(Accession Number: K02159).

SEQ ID NO: 13:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTTATCATTCCAGGTTCCACTGGC

GCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 14: METDTLLLWVLLLWVPGSTG

AEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The USmFZD1crd-hFcm KI chimeric mice expressing a fusion protein of themouse Frizzled 1 extracellular cysteine-rich domain and human Fcm in aB-cell-specific manner are prepared with the use of the pUSmFZD1crd-hFcmKI vector in accordance with the method described in the examples of WO2006/78072.

Further, control chimeric mice used in Example 5 below were prepared inaccordance with the method described in the examples of WO 2006/78072.

Example 5 Analysis of USmFZD1crd-hFcm KI Chimeric Mouse 5-1. NecropsyFinding

The chimeric mice prepared in Example 4 were subjected to necropsy atage of 16 weeks, and the spleen, the liver, the kidney, the adrenalgland, the stomach, the small intestine, the appendix, the largeintestine, the pancreas, the mesenteric lymph node, the female/malereproductive organ, the thymic gland, the lung, the heart, the brain,the muscle, the skin, the femur, the sternum, the cranium, thespondylus, and the costa were observed. As a result, whitening of thefemur, whitening of the sternum, whitening and hardening of the cranium,hardening of the spondylus, and hardening of the costa were observed ascharacteristic changes in the USmFZD1crd-hFcm KI chimeric mice comparedwith the control mice. The number of mice exhibiting changes isdescribed below.

5-1-1. Femur

Whitening was observed more significant in 10 of the 20 USmFZD1crd-hFcmKI chimeric mice subjected to necropsy compared with 10 control mice.

5-1-2. Sternum

Whitening was observed more significant in 18 mice among the 20USmFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with 10control mice.

5-1-3. Cranium

Whitening was observed more significant in 19 mice and hardening wasobserved more significant in 13 mice among the 20 USmFZD1crd-hFcm KIchimeric mice subjected to necropsy compared with 10 control mice.

5-1-4. Spondylus

Hardening was observed more significant in 10 mice among the 20USmFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with 10control mice.

5-1-5. Costa

Hardening was observed more significant in 7 mice among the 20USmFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with 10control mice.

The above results demonstrate that whitening of the femur, whitening ofthe sternum, whitening and hardening of the cranium, hardening of thespondylus, and hardening of the costa may have been induced byoverexpression of the mouse FZD1 extracellular cysteine-richdomain-human Fc mutant fusion constructs.

5-2. Analysis of X-Ray Photograph of Tibia

X-ray photographs (μFX-1000, FUJIFILM) of tibiae obtained from the16-week-old control chimeric mice (9 female mice and 7 male mice) andthe USmFZD1crd-hFcm KI chimeric mice (8 female mice and 3 male mice)were obtained (FIGS. 6 and 7).

In the obtained X-ray photographs of tibiae, whitening was more advancedin both female and male USmFZD1crd-hFcm KI chimeric mice compared withcontrol mice.

The above results demonstrate that whitening of the tibia may have beeninduced by overexpression of mouse FZD1 extracellular cysteine-richdomain-human Fc mutant fusion constructs.

5-3. Blood Cell Analysis

Seventeen 8-week-old USmFZD1crd-hFcm KI female chimeric mice, three8-week-old USmFZD1crd-hFcm KI male chimeric mice, twenty one 8-week-oldfemale control mice and four 8-week-old male control mice, seventeen15-week-old USmFZD1crd-hFcm KI female chimeric mice and three15-week-old USmFZD1crd-hFcm KI male chimeric mice, and seventeen15-week-old female control mice and four 15-week-old male control micewere subjected to orbital blood sampling using a glass capillary underether anesthesia to analyze the blood cell components with the use ofADVIA120 (Bayer Medical Ltd.) (blood cell components; erythrocytecounts, hemoglobin, hematocrit, MCH, MCHC, reticulocyte counts,leukocyte counts, blood platelet counts, lymphocyte counts, neutrophilcounts, monocyte counts, eosinophil counts, and basophil counts). As aresult, the values obtained with the use of the USmFZD1crd-hFcm KIchimeric mice were not significantly different from those of the controlmice at ages of 8 and 15 weeks.

5-4. Pathological Finding

H&E stained pathological sections of the liver, the kidney, the heart,the lung, the spleen, the thymic gland, the mesenteric lymph node, thepancreas, the brain, the adrenal gland, the spermary (in the case ofmale mice), the ovary (in the case of female mice), the femur, thesternum, the stomach, the duodenum, the jejunum, the ileum, theappendix, the colon, the spinal cord, the aorta, the skeletal muscle,and the skin obtained from nine 16-week-old control chimeric mice andtwenty USmFZD1crd-hFcm KI chimeric mice were observed. As a result, thethickened femoral diaphyseal wall (FIGS. 8 and 9, Table 1), theincreased cancellous bone (FIG. 10), and the increased sternalcancellous bone (FIG. 11) were observed as characteristic changes in theUSmFZD1crd-hFcm KI chimeric mice compared with the control mouse. Thenumber of mice exhibiting changes is described below.

TABLE 1 Minimum Maximum Minimum Minimum diaphyseal diaphyseal diaphysealdiaphyseal wall wall wall wall thickness thickness thickness thicknessat site at site at site at site 30% 50% 50% 80% away from away from awayfrom away from Increased proximal Average proximal Average proximalAverage proximal Average cancellous Genes Mouse No.. end (mm) (mm) end(mm) (mm) end (mm) (mm) end (mm) (mm) bone Control TAe1380 0.24 0.250.25 0.31 0.17 0.18 0.15 0.15 − TAe1383 0.25 0.37 0.19 0.15 − mFZD1crd-USN-103FcA2 0.26 0.25 0.36 0.42 0.2 0.20 0.13 0.13 − hFcm USN-103FcA30.28 0.45 0.24 0.16 ± USN-103FcA4 0.24 0.33 0.2 0.09 − USN-103FcB11 0.220.46 0.18 0.13 ++ USN-103FcB14 0.24 0.4 0.21 0.14 + USN-103FcB16 0.240.46 0.18 0.16 + USN-103FcB19 0.25 0.5 0.22 0.07 + Measurement offemoral diaphyseal wall thickness and finding on cancellous bone of16-week-old USmFZD1crd-hFcm KI chimeric mice and control mice(cross-section)

5-4-1. Femur

In comparison with 9 control mice, the thickened diaphyseal wall wasobserved in 11 mice and the increased cancellous bone was observed in 15mice among the 20 USmFZD1crd-hFcm KI chimeric mice subjected tonecropsy. Further, transected sections obtained from 3 femoral sites(i.e., sites 30%, 50%, and 80% away from the proximal end) weresubjected to measurement of the diaphyseal wall thickness using samplesobtained from 7 USmFZD1crd-hFcm KI chimeric mice and 2 control mice. Asa result, the maximum wall thickness of the site 50% away from theproximal end thereof was found to be larger than that of control mice(FIG. 9, Table 1). Also, an increase was observed in the cancellous boneat the site 80% away from the end in 5 of the 7 mice (Table 1).

5-4-2. Sternum

An increase in the cancellous bone was observed more significant in 14mice among the 20 USmFZD1crd-hFcm KI chimeric mice subjected to necropsycompared with 9 control mice.

No significant changes were observed in organs or tissues other thanbones compared with control mice.

The above results demonstrate that the thickened femoral diaphysealwall, the increased cancellous bone, and the increased sternalcancellous bone may have been induced by overexpression of the mouseFZD1 extracellular cysteine-rich domain-human Fc mutant fusionconstructs.

5-5. ELISA Assay Using Serum Sample Aimed at Confirmation of Expressionof Fusion Protein of Mouse FZD1 Extracellular Cysteine-Rich Domain andHuman Fc Mutant in USmFZD1crd-hFcm KI Chimeric Mouse

Fusions of the mouse FZD1 extracellular cysteine-rich domain and thehuman Fc mutant existing in the blood sera of 16-week-oldUSmFZD1crd-hFcm KI chimeric mice (17 female mice and 3 male mice) weredetected via ELISA.

In order to assay the concentration of the fusion protein of the FZD1extracellular cysteine-rich domain and the human Fc mutant in the serumvia ELISA, a test sample or a control sample (Recombinant MouseFrizzled-7/Fc Chimera, R & D Systems, Product Number: 198-FZ; notes:this assay system is a sandwich ELISA system involving the use of anantibody recognizing an Fc region, and use of the mouse Frizzled-7/Fcchimera as a control sample is not considered problematic if expressioncould be confirmed) was applied to a 96-well plate (Maxi Soap, Corning)on which anti-Human IgG (γ-Chain Specific, SIGMA, Product Number: 13382)has been immobilized, incubation was carried out at room temperature for30 minutes, the plate was washed three times with T-PBS(−),peroxidase-labelled antibodies (anti-Human IgG (Fc fragment) peroxidaseconjugates developed in goat, Product Number: A0170, SIGMA) were added,and incubation was then carried out at room temperature for 30 minutes.Thereafter, the plate was washed four times with T-PBS(−), a color wasdeveloped using a Sumilon peroxidase color-developing kit (ProductNumber: ML-1120T, Sumitomo Bakelite Co. Ltd.), and the absorbance at 450nm was assayed to determine the concentration in the serum.

As a result, the average concentration among 17 female mice was 298.4μg/ml, that among 3 male mice was 308.8 μg/ml (both values arereferences), and the concentrations assayed with the use of the serumsamples obtained from 5 female control mice and a male control mousewere lower than the detection limit.

The above results suggest that the fusion protein of the mouse FZD1extracellular cysteine-rich domain and the human Fc mutant is expressedin vivo and circulated in the blood.

Example 6 Preparation of UShFZD1crd-hFcm KI Chimeric Mouse

A pUShFZD7crd-hFcm KI vector was prepared from human FZD1-cDNA (SEQ IDNO: 15) and human IgG1 Fc mutant-cDNA (SEQ ID NO: 3) in accordance withthe method described in Example 1.

The human FZD1 signal sequence, a CRD (the cystein-rich-domain), and aregion located downstream of a CRD comprising the 7-transmembrane domainin SEQ ID NO: 15 are marked by a single underline, a solid box, and adouble underline, respectively, based on the information regarding theGenBank Accession Numbers: NM_003505.1 and NP_003496.1.

SEQ ID NO: 15:ATGGCTGAGGAGGAGGCGCCTAAGAAGTCCCGGGCCGCCGGCGGTGGCGCGAGCTGGGAACTTTGTGCCGGGGCGCTCTCGGCCCGGCTGGCGGAGGAGGGCAGCGGGGACGCCGGTGGCCGCCGCCGCCCGCCAGTTGACCCCCGGCGATTGGCGCGCCAGCTGCTGCTGCTGCTTTGGCTGCTGGAGGCTCCGCTGCTGCTGGGGGTCCGGGCC

GGCGGCGGAGGGCACCGTGGCGGCTTCCCGGGGGGCGCCGGCGCGTCGGAGCGAGGCAAGTTCTCCTGCCCGCGCGCCCTCAAGGTGCCCTCCTACCTCAACTACCACTTCCTGGGGGAGAAGGACTGCGGCGCACCTTGTGAGCCGACCAAGGTGTATGGGCTCATGTACTTCGGGCCCGAGGAGCTGCGCTTCTCGCGCACCTGGATTGGCATTTGGTCAGTGCTGTGCTGCGCCTCCACGCTCTTCACGGTGCTTACGTACCTGGTGGACATGCGGCGCTTCAGCTACCCGGAGCGGCCCATCATCTTCTTGTCCGGCTGTTACACGGCCGTGGCCGTGGCCTACATCGCCGGCTTCCTCCTGGAAGACCGAGTGGTGTGTAATGACAAGTTCGCCGAGGACGGGGCACGCACTGTGGCGCAGGGCACCAAGAAGGAGGGCTGCACCATCCTCTTCATGATGCTCTACTTCTTCAGCATGGCCAGCTCCATCTGGTGGGTGATCCTGTCGCTCACCTGGTTCCTGGCGGCTGGCATGAAGTGGGGCCACGAGGCCATCGAAGCCAACTCACAGTATTTTCACCTGGCCGCCTGGGCTGTGCCGGCCATCAAGACCATCACCATCCTGGCGCTGGGCCAGGTGGACGGCGATGTGCTGAGCGGAGTGTGCTTCGTGGGGCTTAACAACGTGGACGCGCTGCGTGGCTTCGTGCTGGCGCCCCTCTTCGTGTACCTGTTTATCGGCACGTCCTTTCTGCTGGCCGGCTTTGTGTCGCTCTTCCGCATCCGCACCATCATGAAGCACGATGGCACCAAGACCGAGAAGCTGGAGAAGCTCATGGTGCGCATTGGCGTCTTCAGCGTGCTGTACACTGTGCCAGCCACCATCGTCATCGCCTGCTACTTCTACGAGCAGGCCTTCCGGGACCAGTGGGAACGCAGGTGGGTGGCCCAGAGCTGCAAGAGCTACGCTATCCCCTGCCCTCACCTCCAGGCGGGCGGAGGCGCCCCGCCGCACCCGCCCATGAGCCCGGACTTCACGGTCTTCATGATTAAGTACCTTATGACGCTGATCGTGGGCATCACGTCGGGCTTCTGGATCTGGTCCGGCAAGACCCTCAACTCCTGGAGGAAGTTCTACACGAGGCTCACCAACAGCAAACAAGGGGAGACTACAGTCTGA

The amino acid sequence encoded by SEQ ID NO: 15 (574 amino acids, SEQID NO: 16) is shown below.

SEQ ID NO: 16:MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWLLEAPLLLGVRA

GGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYGLMYFGPEELRFSRTWIGIWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYTAVAVAYIAGFLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVGLNNVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTYPATIVIACYFYEQAFRDQWERSWVAQSCKSYAIPCPHLQAGGGAPPHPPMSPDFTVFMIKYLMTLIVGITSGFWIWSGKTLNSWRKFYTRLTNSKQGETTV

A polynucleotide sequence comprising a region from the initiation codonto the termination codon of the pUShFZD1crd-hFcm KI vector expressionunit (SEQ ID NO: 17; a 1,546-bp sequence comprising a mouse Igκ signalsequence containing an intron region (a region marked by a singleunderline) substituted with the human FZD1 signal sequence and the humanFZD1crd-hFcm sequence located downstream thereof; wherein the regionmarked by a solid box represents the human Frizzled 1 extracellularcysteine-rich domain and the region marked by a double underlinerepresents hFcm) and the amino acid sequence encoded by the cDNA (SEQ IDNO: 18; a sequence comprising 434 amino acids; wherein the region markedby a single underline represents the mouse Igκ signal sequence, theregion marked by a solid box represents the human Frizzled 1extracellular cysteine-rich domain, and the region marked by a doubleunderline represents hFcm) are shown below. Information regarding themouse Igκ signal sequence containing an intron region was obtained fromthe UCSC mouse genome database as the genome sequence located upstreamof MUSIGKVR1 obtained from the GenBank (Accession Number: K02159).

SEQ ID NO: 17:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTTATCATTCCAGGTTCCACTGGC

GCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 18: METDTLLLWVLLLWVPGSTG

AEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The UShFZD1crd-hFcm KI chimeric mice expressing a fusion protein of thehuman Frizzled 1 extracellular cysteine-rich domain and human Fcm in aB-cell-specific manner are prepared with the use of the pUShFZD1crd-hFcmKI vector in accordance with the method described in the examples of WO2006/78072.

Control chimeric mice into which no foreign cDNA expression unit hasbeen inserted are prepared in accordance with the method described inthe examples of WO 2006/78072.

Example 7

Expression and Preparation of mFZD7crd-hFcm Recombinant7-1. Construction of mFZD7crd-hFcm Recombinant Expression Vector7-1-1. Construction of pLN1V5 Vector

Sense oligo DNA (V5S) having the BamHI, NheI, and SalI sites at the 5′terminus and the XhoI site at the 3′ terminus (a V5 tag and a stopcodon) and corresponding antisense oligo DNA (V5AS) were synthesized.

V5S: (SEQ ID NO: 50) GATCCGCTAGCGTCGACGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTGAC V5AS: (SEQ ID NO: 51)TCGAGTCACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACC GTCGACGCTAGCG

Oligo DNA synthesized above was introduced into the BamHI-XhoI site onthe pLN1 vector described in the report of Kakeda et al. (Gene Ther.,12, 852-856, 2005) to construct the pLN1V5 vector.

7-1-2. Synthesis of mFZD7crd-hFcm DNA Fragment

088Fc_BHIkozakFw: (SEQ ID NO: 52)TAAAGGATCCCGGCCACCATGCGGGGCCCCGGCACGGCGG 088Fc_mFZD7G1SA_3 primer:(SEQ ID NO: 53) GTCTGAAGACCTAGGCTCGGCCAGGTAGGGAGCAGTAGGG G1SA_5primer: (SEQ ID NO: 54) GCCGAGCCTAGGTCTTCAGAC SaLIG1SARev:  (SEQ ID NO: 55)TAAAGTCGACTCATTTACCCGGAGACAGGG

A reaction solution was prepared using Prime STAR HS DNA Polymerase(Takara Bio Inc., Japan) in accordance with the instructions, 10 pmoleach primers shown in SEQ ID NOs: 52 and 53 and mouse FZD7 cDNA (SEQ IDNO: 1) as a template were added to 50 μl of the reaction solution, theresultant was incubated at 98° C. for 1 minute, an amplification cycleof 98° C. for 10 seconds, 57° C. for 5 seconds, and 72° C. for 2 minuteswas repeated 20 times, and the resulting 594-bp amplified fragment wasseparated and recovered with 0.8% gel. The amplified fragment (BamHImFZD7crd hFcm) was recovered from the gel using the QIAquick GelExtraction Kit (Qiagen, Japan) in accordance with the instructions.

Similarly, a reaction solution was prepared using Prime STAR HS DNAPolymerase (Takara Bio Inc., Japan) in accordance with the instructions,10 pmol each primers shown in SEQ ID NOs: 54 and 55 and hFcm cDNA (SEQID NO: 3) as a template were added to 50 μl of the reaction solution,the resultant was incubated at 98° C. for 1 minute, an amplificationcycle of 98° C. for 10 seconds, 57° C. for 5 seconds, and 72° C. for 2minutes was repeated 20 times, and the resulting 712-bp amplifiedfragment was separated and recovered with 0.8% gel. The amplifiedfragment (hFcm SalI) was recovered from the gel using the QIAquick GelExtraction Kit (Qiagen, Japan) in accordance with the instructions.

The amplified DNA fragments obtained via the two above PCR procedures(i.e., BamHI mFZD7 hFcm and hFcm SalI) were added to the PrimeSTARbuffer to bring the total amount to 100 μl, the solution was heated at100° C. for 10 minutes, and the temperature was reduced to roomtemperature, followed by annealing of the hFcm region. Thereafter, 10pmol each primers shown in SEQ ID NOs: 52 and 55 were added, anextension reaction was carried out at 72° C. for 5 minutes, anamplification cycle of 98° C. for 10 seconds, 57° C. for 5 seconds, and72° C. for 2 minutes was repeated 20 times, incubation was carried outat 72° C. for 2 minutes in the end, and the resulting 1,285-bp amplifiedfragment was separated and recovered with 0.8% gel. The amplifiedfragment was recovered from the gel using the QIAquick Gel ExtractionKit (Qiagen, Japan) in accordance with the instructions.

7-1-3. Construction of mFZD7crd-hFcm Recombinant Expression Vector

The PCR-amplified fragment recovered in Example 7-1-2 was digested withthe BamHI and SalI restriction enzymes (Roche Diagnostics, K. K.,Japan), and the resultant was separated and recovered with 0.8% agarosegel. The enzyme-treated fragment was recovered from the gel using theQIAquick Gel Extraction Extraction Kit (Qiagen, Japan) in accordancewith the instructions. The obtained enzyme-treated fragment wasintroduced into the BamHI.SalI site of the pLN1V5 vector prepared inExample 7-1-1 to construct the mFZD7crd-hFcm recombinant expressionvector (FIG. 12).

A polynucleotide sequence (1,257 bp, SEQ ID NO: 56) comprising a regionfrom the initiation codon to the termination codon of mFZD7crd-hFcmrecombinant cDNA and the amino acid sequence (418 amino acids, SEQ IDNO: 57) comprising a signal sequence of mFZD7-hFcm encoded by the cDNAare shown below. In SEQ ID NOs: 56 and 57, an underlined portionrepresents the mouse FZD7 signal sequence.

SEQ ID NO: 56: ATGCGGGGCCCCGGCACGGCGGCGTCGCACTCGCCCCTGGGCCTCTGCGCCCTGGTGCTTGCTCTTCTGTGCGCGCTGCCCACGGACACCCGGGCTCAGCCATATCACGGCGAGAAAGGCATCTCGGTACCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGTTGTGCACGGATATCGCCTACAACCAGACCATCCTGCCCAACCTGCTGGGCCACACGAACCAAGAGGACGCGGGCCTCGAGGTGCACCAGTTCTACCCTCTGGTAAAGGTGCAGTGTTCTCCTGAGCTACGCTTCTTCTTATGCTCTATGTACGCACCCGTGTGCACCGTGCTCGACCAAGCCATTCCTCCGTGCCGTTCCTTGTGCGAGCGCGCCCGACAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGAGCGGTTGCGCTGCGAGAACTTCCCAGTGCACGGTGCCGGCGAGATCTGCGTGGGGCAGAACACGTCCGACGGCTCCGGGGGCGCGGGCGGCAGTCCCACCGCCTACCCTACTGCTCCCTACCTGGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAATGASEQ ID NO: 57: MRGPGTAASHSPLGLCALVLALLCALPTDTRAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGAGGSPTAYPTAPYLAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK7-2. Transient Expression of mFZD7crd-hFcm Using mFZD7crd-hFcmRecombinant Expression Vector

7-2-1. Preparation of Expression Vector Used for Gene Introduction

The mFZD7crd-hFcm recombinant expression vector obtained in Example7-1-3 was introduced into E. coli DH5a, and DNA was prepared from thetransformant using a plasmid purification kit (Qiagen plasmid Maxi kit,Qiagen, Japan).

7-2-2. Introduction of Vector into Cultured Cell and SecretoryExpression

FreeStyle 293F cells (Invitrogen Japan K. K.) are cultured in FreeStyle293 expression medium (Invitrogen Japan K. K.) at 37° C. in the presenceof 5% CO₂ at 125 rpm to reach a cell density of 2×10⁵ to 3×10⁶ cells/ml.When culture was conducted using 1 liter of medium, a solutioncomprising 35 ml of the Opti-MEM I reduced serum medium (InvitrogenJapan K. K.) added to 1 mg of the expression vector and a solutioncomprising 33.7 ml of the Opti-MEM I reduced serum medium added to 1.3ml of the 293 fectin transfection reagent (Invitrogen Japan K. K.) wereprepared, and the resulting solutions were incubated at room temperaturefor 5 minutes. These solutions were mixed with each other afterincubation, and the resultant was incubated at room temperature for anadditional about 30 minutes. Thereafter, the expression vector treatedin the manner described above was added to a medium containing 1×10⁹cells/1 of FreeStyle 293F cells, and culture was conducted for 3 days.

7-3. Purification and Preparation of mFZD7crd-hFcm Recombinant

7-3-1. Pretreatment of Culture Supernatant

The supernatant of the culture solution obtained in Example 7-2-2 wasrecovered, filtered through a 0.22 μm filter (0.22 μm GP ExpressMembrane 500 ml, Millipore, Japan), and then cooled to 4° C.

7-3-2. Antibody Affinity Chromatography

The acidic buffer used is 1 liter of a solution comprising 3.895 g ofcitrate monohydrate (Nakalai Tesque, Inc., Japan), 0.38 g of trisodiumcitrate (Wako Pure Chemical Industries, Ltd., Japan), and 2.92 g ofsodium chloride (Junsei Chemical Co., Ltd., Japan) dissolved in water.The neutralizing buffer used is 1 liter of a solution comprising 13.1 gof sodium dihydrogen phosphate dihydrate (Kanto Chemical Co., Inc.,Japan) and 41.5 g of disodium hydrogen phosphate dodecahydrate (WakoPure Chemical Industries, Ltd., Japan) dissolved in water.

The pretreated culture supernatant (1 liter) was applied to aPBS-equilibrated protein G column (Hi Trap Protein G HP, 5 ml,GEHealthcare Bio-Sciences Corp., Japan). Thereafter, the column waswashed with 25 ml or more PBS, then with 25 ml or more buffer preparedby adding NaCl to PBS to bring the NaCl concentration to 1.85 M, andwith 30 ml of PBS again. After the completion of the washing procedure,25 ml of acidic buffer was added to the column, and the target proteinwas recovered. The target protein was neutralized with a neutralizingbuffer immediately after it was recovered. AKTAexplorer 10s (GEHealthcare Bio-Sciences Corp, Japan) was used in the separation andpurification procedure. Endotoxin was removed before use.

7-3-3. Preparation of Purified Authentic Sample

The purified authentic sample obtained in Example 7-3-2 was concentratedusing an ultrafilter membrane VIVASPIN20 10,000 MWCO PES (SartoriusStedim Japan K. K., Japan). Thereafter, the buffer in the sample wassubstituted with PBS using NAP-25 Columns (GE Healthcare Bio-SciencesCorp, Japan). After the completion of the concentration and substitutionprocedure, the resultant was filtered through a 0.22 μm filter (Millex GV, Millipore, Japan).

A protein concentration was determined by measuring the specificabsorbance at 280 nm (A280 nm) (E1%, 1 cm=10.3).

Example 8

Analysis of Mouse to which mFZD7crd-hFcm Recombinant has beenAdministered

8-1. Administration to Mouse

The mFZD7crd-hFcm recombinant was administered to mice in order toevaluate physiological effects thereof on bone tissue.

Since the mFZD7crd-hFcm recombinant is a protein comprising the humanantibody Fc region, the possibility of suppression of activity of themFZD7crd-hFcm recombinant upon production of the neutralizing antibodyin the body resulting from administration was considered. Thus, fullyhuman antibody-producing mice (JP Patent No. 3523245) were used for theadministration experiment in order to reduce a risk for the productionof neutralizing antibodies. The mice were introduced and naturalized atage of 3 weeks, 1 μl of the blood was sampled from the caudal vein atage of 4 weeks, the blood was dispensed in a 96-well plate (NuncImmunoplateII 96 Maxi Soap 442404, Thermo Fisher Scientific K. K.,Japan) on which the anti-human IgG (γ-chain specific) goat antibodies(Product Number: 13382, Sigma-Aldrich Japan K. K.) were immobilized,human IgG in the blood was solid-phased, and human antibody productionin the mouse blood was assayed via ELISA involving the use of anti-humanIgG (Fc fragment) peroxidase labelled goat antibodies (Product Number:A0170, Sigma-Aldrich Japan K. K.) as detection antibodies and developinga color using a Sumilon peroxidase color-developing kit T (ProductNumber: ML-1120T, Sumitomo Bakelite Co. Ltd., Japan). Mice in whichhuman antibody production was observed in the blood were divided intogroups based on body weights at age of 6 weeks on the previous day ofthe initiation of administration (i.e., day 1).

The mFZD7crd-hFcm recombinant was diluted with PBS to adjust a proteinconcentration to 5 mg/ml, and the resultant was administered into thecaudal veins of mice of the mFZD7crd-hFcm recombinant test group inamounts of 200 μl per mouse once every 10 days (seven times in total).As a control group for comparison of changes in bone tissue, anon-treatment group was designated. The day of the initialadministration was designated as day 0, the recombinant was administeredto the caudal vein every 10 days up to day 60 (seven times in total),and mice were subjected to necropsy on day 68.

8-2. Pathological Finding

Tissues were sampled from the right femur, sternum, and the like atnecropsy, the tissue samples were soaked and fixed in a 10% neutralbuffered formalin solution (Wako Pure Chemical Industries, Ltd., Japan),the tissue samples were demineralized, H&E samples were prepared, andthe maximum diaphyseal wall thickness at a site 50% away from theproximal end of the femur was measured.

The diaphyseal wall thickness of the samples obtained from 5 controlmice and 4 mice to which the mFZD7crd-hFcm recombinants had beenadministered were measured at necropsy. As a result, the maximumdiaphyseal wall thickness at a site 50% away from the proximal end ofmice of the test group was higher than that of the control group (Table2).

TABLE 2 Maximum diaphyseal wall Animal thickness at site 50% awayAverage Groups No. from proximal end (mm) (mm) Control INT52 0.32 0.35INT53 0.42 INT54 0.39 INT55 0.33 INT56 0.28 mFZD7crd-hFcm mF7FcA7 0.320.44 mF7FcA9 0.50 mF7FcA10 0.47 mF7FcA11 0.45 Measurement of diaphysealwall thickness of femurs (cross sections) of mice to which mFZD7crd-hFcmrecombinants have been administered and control mice

8-3. Necropsy Finding

The femurs, sternums, and craniums of 4 mice to which the mFZD7crd-hFcmrecombinants had been administered in Example 8-2 were observed atnecropsy. As a result, whitening of the femur, whitening of the sternum,whitening of the cranium, whitening of the sternum, and a tendency ofthickening node were observed as characteristic changes in mice to whichthe mFZD7crd-hFcm recombinants had been administered. The number of miceexhibiting changes is described below.

8-3-1. Femur

Whitening was observed in 3 mice among the 4 mice subjected to necropsyto which the mFZD7crd-hFcm recombinants had been administered.

8-3-2. Cranium

Whitening was observed in 2 mice among the 4 mice subjected to necropsyto which the mFZD7crd-hFcm recombinants had been administered.

8-3-3. Sternum

Whitening was observed in a mouse among the 4 mice subjected to necropsyto which the mFZD7crd-hFcm recombinants had been administered, and atendency of thickening node was observed in 2 mice among such mice.

The above results suggest the possibility that whitening of the femur,whitening of the cranium, whitening of the sternum, and a tendency ofthickening node were induced by administration of the mFZD7crd-hFcmrecombinants.

8-4. Bone Morphometry

Tibial tissues were sampled at necropsy, samples of undemineralizedtibial sections were prepared, and the resulting samples were subjectedto toluidine blue staining. In order to prepare section samples, thetibia samples were embedded in GMA (glycolmethacrylate) resin inadvance. The metaphyseal secondary cancellous bones of the obtainedsamples of undemineralized sections were subjected to measurement of thebone volume/tissue volume (BV/TV) as the structural parameter.

As a result of the measurement of the bone volume/tissue volume (BV/TV)of samples obtained from 5 control mice and 4 mice to which themFZD7crd-hFcm recombinants had been administered, the increased BV/TVwas observed at necropsy in the group to which the mFZD7-hFcrecombinants had been administered compared with the control group.Accordingly, the increased bone volume/tissue volume was considered tohave been induced by administration of the mFZD7crd-hFcm recombinants inthe secondary cancellous bone of the tibial metaphysis (Table 3).

TABLE 3 Animal Groups No. BV/TV (%) Average (%) Control INT52 11.06 9.30INT53 8.96 INT54 5.60 INT55 15.95 INT56 4.94 mFZD7crd-hFcm mF7FcA7 14.9416.69 mF7FcA9 20.26 mF7FcA10 19.24 mF7FcA11 12.30 Bone volume/tissuevolume of secondary cancellous bone region of mice to whichmFZD7crd-hFcm recombinants have been administered and that of tibialmetaphyseal end of control mice

Example 9 Preparation of USmFZD2crd-hFcm KI Chimeric Mouse

A pUSmFZD2crd-hFcm KI vector was prepared from mouse FZD2-cDNA (a1,713-bp sequence comprising a region from an initiation codon to atermination codon, SEQ ID NO: 58) and human IgG1 Fc mutant-cDNA (SEQ IDNO: 3) in accordance with the method described in Example 1.

The mouse FZD2 signal sequence, a CRD (the cystein-rich-domain), and aregion located downstream of a CRD comprising the 7-transmembrane domainin SEQ ID NO: 58 are marked by a single underline, a solid box, and adouble underline, respectively, based on the information regarding theGenBank Accession Numbers: NM_020510.2 and NP_065256.1.

SEQ ID NO: 58:ATGCGGGCCCGCAGCGCCCTGCCCCGCAGCGCCCTGCCCCGCCTGCTGCTGCCACTGCTGCTGCTGCCGGCCGCCGGACCGGCC

CTCACCACCGCGCCACCTTCTGGGCTGCAGCCCGGCGCGGGTGGCACCCCGGGCGGCCCTGGCGGTGGTGGCTCGCCACCGCGTTACGCCACTCTGGAGCACCCTTTCCACTGTCCCCGCGTCCTCAAGGTGCCGTCCTATCTCAGCTATAAGTTTCTGGGTGAGCGCGATTGTGCCGCGCCCTGCGAGCCCGCACGGCCCGACGGCTCTATGTTCTTCTCGCAAGAGGAGACTCGTTTTGCCCGTCTCTGGATCCTCACATGGTCGGTGTTGTGCTGCGCTTCCACTTTCTTCACGGTCACCACCTATTTAGTGGACATGCAGCGATTTCGCTACCCAGAGCGGCCCATCATCTTTCTGTCCGGCTGCTACACCATGGTGTCAGTGGCCTACATTGCGGGCTTCGTTCTCCAGGAGCGCGTGGTATGCAATGAGCGCTTCTCAGAGGACGGTTATCGCACGGTGGTGCAGGGCACTAAGAAAGAAGGCTGCACTATACTCTTCATGATGCTCTACTTCTTCAGCATGGCCAGCTCCATCTGGTGGGTGATTCTGTCCCTCACCTGGTTCCTGGCAGCCGGAATGAAGTGGGGCCACGAGGCCATCGAGGCCAATTCGCAGTACTTCCACCTGGCCGCCTGGGCCGTGCCGGCCGTCAAAACCATCACCATCTTGGCCATGGGCCAGATCGACGGCGACCTGCTGAGCGGCGTGTGCTTCGTGGGCCTCAATAGCCTGGACCCGCTGCGGGGCTTCGTGCTGGCGCCGCTCTTCGTATACCTGTTCATCGGTACATCCTTCCTGCTGGCCGGCTTCGTGTCACTCTTCCGCATCCGCACCATCATGAAGCACGACGGCACCAAGACGGAGAAGCTGGAGAGGCTCATGGTGCGCATTGGCGTCTTCTCGGTGCTCTACACGGTACCGGCCACCATCGTCATCGCCTGCTACTTCTATGAGCAGGCCTTCCGCGAGCACTGGGAGCGCTCCTGGGTAAGCCAGCACTGCAAGAGCCTAGCCATCCCCTGCCCGGCCCACTACACGCCCCGCATGTCGCCCGACTTCACAGTCTACATGATCAAATACCTCATGACGCTCATCGTGGGCATCACGTCGGGCTTCTGGATCTGGTCCGGCAAGACACTGCACTCGTGGAGGAAGTTCTACACTCGTCTCACCAACAGCCGGCATGGCGAGACCACTGTGTGA

The amino acid sequence encoded by SEQ ID NO: 58 (570 amino acids, SEQID NO: 59) is shown below.

SEQ ID NO: 59: MRARSALPRSALPRLLLPLLLLPAAGPA

LTTAPPSGLQPGAGGTPGGPGGGGSPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEETRFARLWILTWSVLCCASTFFTVTTYLVDMQRFRYPERPIIFLSGCYTMVSVAYIAGFVLQERVVCNERFSEDGYRTVVQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAVKTITILAMGQIDGDLLSGVCFVGLNSLDPLRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLERLMVRIGVFSVLYTVPATIVIACYFYEQAFREHWERSWVSQHCKSLAIPCPAHYTPRMSPDFTVYMIKYLMTLIVGITSGFWIWSGKTLHSWRKFYTRLTNSRHGETTV

A polynucleotide sequence comprising a region from the initiation codonto the termination codon of the pUSmFZD2crd-hFcm KI vector expressionunit (SEQ ID NO: 60; a 1,423-bp sequence comprising a mouse Igκ signalsequence containing an intron region (a region marked by a singleunderline) substituted with the mouse FZD2 signal sequence and the mouseFZD2crd-hFcm sequence located downstream thereof; wherein the regionmarked by a solid box represents the mouse Frizzled 2 extracellularcysteine-rich domain, and the region marked by a double underlinerepresents hFcm) and the amino acid sequence encoded by the cDNA (SEQ IDNO: 61; a sequence comprising 393 amino acids; wherein the region markedby a single underline represents the mouse Igκ signal sequence, theregion marked by a solid box represents the mouse Frizzled 2extracellular cysteine-rich domain, and the region marked by a doubleunderline represents hFcm) are shown below. Information regarding themouse Igκ signal sequence containing an intron region was obtained fromthe UCSC mouse genome database as the genome sequence located upstreamof MUSIGKVR1 obtained from the GenBank (Accession Number: K02159).

SEQ ID NO: 60:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATAT AACAATAATCTTTGTGTTTATCATTCCAGGTTCCACTGGC

GCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 61: METDTLLLWVLLLWVPGSTG

AEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The USmFZD2crd-hFcm KI chimeric mice expressing a fusion protein of themouse FZD2 extracellular cysteine-rich domain and human Fcm in aB-cell-specific manner are prepared with the use of the pUSmFZD2crd-hFcmKI vector in accordance with the method described in the Examples of WO2006/78072.

Since the amino acid sequence of the FZD2 extracellular cysteine-richdomain of a human is identical to that of a mouse, the USmFZD2crd-hFcmKI chimeric mouse is substantially identical to the UShFZD2crd-hFcm KIchimeric mouse expressing a fusion protein of the human FZD2extracellular cysteine-rich domain and human Fcm.

Further, control chimeric mice used in Example 10 were prepared inaccordance with the method described in Example 11 of WO 2006/78072.

Example 10 Analysis of USmFZD2crd-hFcm KI Chimeric Mouse 10-1. NecropsyFinding

The chimeric mice prepared in Example 9 were subjected to necropsy atage of 16 weeks, and the spleen, the liver, the kidney, the adrenalgland, the stomach, the small intestine, the appendix, the largeintestine, the pancreas, the mesenteric lymph node, the female/malereproductive organ, the thymic gland, the lung, the heart, the brain,the muscle, the skin, the femur, the sternum, the cranium, thespondylus, and the costa were observed. As a result, whitening of thefemur, whitening of the sternum, whitening and hardening of the cranium,hardening of the spondylus, and hardening of the costa were observed ascharacteristic changes in the USmFZD2crd-hFcm KI chimeric mice comparedwith the control mice. The number of mice exhibiting changes isdescribed below.

10-1-1. Femur

Whitening was observed more significant in 7 mice among the 12USmFZD2crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice.

10-1-2. Sternum

Whitening was observed more significant in 9 mice among the 12USmFZD2crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice.

10-1-3. Cranium

Whitening was observed more significant in 6 mice and hardening wasobserved more significant in 4 mice among the 12 USmFZD1crd-hFcm KIchimeric mice subjected to necropsy compared with 6 control mice.

10-1-4. Spondylus

Hardening was observed more significant in 2 mice among the 12USmFZD2crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice.

10-1-5. Costa

Hardening was observed more significant in 2 mice among the 12USmFZD2crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice.

The above results demonstrate that whitening of the femur, whitening ofthe sternum, whitening and hardening of the cranium, hardening of thespondylus, and hardening of the costa that are considered to result fromthe increased bone mass may have been induced by overexpression of thefusion protein of mouse FZD2 extracellular cysteine-rich domain andhuman Fc mutant.

10-2. Pathological Finding

In accordance with the method described in Example 8-2, the maximumdiaphyseal wall thickness at sites 30%, 50%, and 80% away from theproximal end of the femur was measured.

Diaphyseal wall thickness of samples obtained from 6 control mice andfrom 12 USmFZD2crd-hFcm KI chimeric mice were measured at necropsy. As aresult, the maximum diaphyseal wall thickness at a site 50% away fromthe proximal end and the minimum diaphyseal wall thickness at a site 30%away therefrom were larger than those of control samples (Table 4).

TABLE 4 Minimum Maximum Minimum Minimum diaphyseal diaphyseal diaphysealdiaphyseal wall thickness wall thickness wall thickness wall thicknessat site 30% at site 50% at site 50% at site 80% away from away from awayfrom away from proximal end Average proximal end Average proximalAverage proximal end Average Gene Name Mouse ID No. (mm) (mm) (mm) (mm)end (mm) (mm) (mm) (mm) Control TAe1943 0.20 0.20 0.26 0.36 0.16 0.180.11 0.12 TAe1945 0.20 0.31 0.20 0.16 TAe1947 0.22 0.32 0.18 0.12TAe1949 0.20 0.44 0.18 0.10 TAe1950 0.20 0.40 0.17 0.11 TAe1951 0.200.40 0.17 0.11 mFZD2crd- USN-155FcA1 0.25 0.24 0.38 0.43 0.19 0.19 0.130.12 hFcm USN-155FcA2 0.23 0.30 0.20 0.15 USN-155FcA3 0.21 0.38 0.210.14 USN-155FcA4 0.23 0.40 0.20 0.11 USN-155FcA5 0.26 0.42 0.18 0.10USN-155FcA6 0.25 0.53 0.13 0.13 USN-155FcB7 0.26 0.42 0.21 0.10USN-155FcB9 0.22 0.49 0.20 0.12 USN-155FcB10 0.22 0.46 0.17 0.11USN-155FcB11 0.29 0.45 0.22 0.11 USN-155FcB12 0.24 0.48 0.20 0.09USN-155FcB16 0.24 0.40 0.21 0.10 Measurements of diaphyseal wallthickness of femers (cross sections) of 16-week-old USmFZD2crd-hFcm KIchimeric mice and control mice

The above results demonstrate that the thickened femoral diaphyseal wallmay have been induced by overexpression of the fusion protein of mouseFZD2 extracellular cysteine-rich domain and human Fc mutant.

Example 11

Expression and Preparation of Recombinant mFZD1crd-hFcm11-1. Construction of Recombinant mFZD1crd-hFcm Expression Vector

In accordance with the method described in Example 7-1, the recombinantmFZD1crd-hFcm expression vector was constructed using the PCR primersshown in SEQ ID NOs: 54, 55, 62, and 63 and, as templates, mouse Fzd1cDNA (SEQ ID NO: 11) and hFcm cDNA (SEQ ID NO: 3) (FIG. 13).

103Fc_BHIkozakFw:  (SEQ ID NO: 62)TAAA GGATCCCGGCCACC ATGGCTGAGGAGGCGGCGCC 103Fc_mFZD1G1SA_3 primer:(SEQ ID NO: 63) GTCTGAAGACCTAGGCTCGGC GTGCTGCGGATTACTGGTCC

A polynucleotide sequence comprising a region from the initiation codonto the termination codon of recombinant mFZD1crd-hFcm cDNA (1,446 bp,SEQ ID NO: 64) and an amino acid sequence comprising a signal sequenceof mFZD1-hFcm encoded by the cDNA (481 amino acids, SEQ ID NO: 65) areshown below. In SEQ ID NOs: 64 and 65, the underlined portion representsthe signal sequence of mouse FZD1.

SEQ ID NO: 64: ATGGCTGAGGAGGCGGCGCCTAGCGAGTCCCGGGCCGCCGGCCGGCTGAGCTTGGAACTTTGTGCCGAAGCACTCCCGGGCCGGCGGGAGGAGGTGGGGCACGAGGACACGGCCAGCCACCGCCGCCCCCGGGCTGATCCCCGGCGTTGGGCTAGCGGGCTGCTGCTGCTGCTTTGGTTGCTGGAGGCTCCTCTGCTTTTGGGGGTCCGAGCGCAGGCGGCGGGCCAGGTATCCGGGCCGGGCCAGCAAGCCCCGCCGCCGCCCCAGCCCCAGCAGAGCGGGCAGCAGTACAACGGCGAACGGGGCATCTCCATCCCGGACCACGGCTACTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAATCAGGAGGACGCCGGTCTGGAGGTGCACCAGTTCTACCCTCTGGTGAAGGTGCAGTGCTCCGCCGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCTGTGTGCACCGTACTGGAGCAGGCGCTACCGCCCTGCCGCTCCCTGTGCGAGCGCGCACGCCAGGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAGTGGCCAGACACACTCAAGTGCGAGAAGTTCCCGGTGCACGGCGCAGGAGAGCTGTGCGTGGGCCAGAACACGTCCGACAAAGGCACCCCAACTCCCTCCTTGCTACCAGAGTTCTGGACCAGTAATCCGCAGCACGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 65:MAEEAAPSESRAAGRLSLELCAEALPGRREEVGHEDTASHRRPRADPRRWASGLLLLLWLLEAPLLLGVRAQAAGWVGPGQQAPPPPQPQQSGQQYNGERHGISIDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVIINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK11-2. Transient Expression of Recombinant mFZD1crd-hFcm UsingRecombinant mFZD1crd-hFcm Expression Vector

11-2-1. Preparation of Expression Vector Used for Gene Introduction

The recombinant mFZD1crd-hFcm expression vector obtained in Example 11-1was introduced into E. coli DH5a, and DNA was prepared from thetransformant cells using a plasmid purification kit (Qiagen plasmid Maxikit; Qiagen, Japan).

11-2-2. Introduction of Vector into Cultured Cell and SecretoryExpression

FreeStyle 293F cells (Invitrogen, Japan) are cultured in FreeStyle 293expression medium (Invitrogen, Japan) at 37° C. in the presence of 5%CO₂ at 125 rpm to reach a cell density of 2×10⁵ to 3×10⁶ cells/ml. Whenculture was conducted using 1 liter of medium, 20 ml of Opti PRO SFM(Invitrogen, Japan) was added to 1 mg of the expression vector, and 17.5ml of Opti PRO SFM was added to 2.5 ml of PEI (polyethylenimine). Thesesolutions were mixed with each other immediately thereafter, and theresultant was incubated at room temperature for 10 minutes. Thereafter,the expression vector treated in the manner described above was added toa medium containing 1×10⁹ cells/1 of FreeStyle 293F cells, and culturewas conducted for 3 days.

11-3. Purification and Preparation of mFZD1crd-hFcm Recombinant

11-3-1. Pretreatment of Culture Supernatant

The supernatant of the culture solution obtained in Example 11-2-2 wasrecovered, the supernatant was filtered through a 0.22 μm filter (0.22μm GP Express Membrane 500 ml; Millipore, Japan) and then cooled to 4°C. (in a cold room).

11-3-2. Antibody Affinity Chromatography

The acidic buffer used is 1 liter of a solution comprising 3.895 g ofcitrate monohydrate (Nakalai Tesque, Inc., Japan, MW: 210.14), 0.38 g oftrisodium citrate (Wako Pure Chemical Industries, Ltd., Japan, MW:258.07), and 2.92 g of sodium chloride (Junsei Chemical Co., Ltd.,Japan, MW: 58.44) dissolved in Milli-Q water. The neutralizing bufferused is 1 liter of a solution comprising 13.1 g of sodium dihydrogenphosphate dihydrate (Kanto Chemical Co., Inc., MW: 156.01), 41.5 g ofdisodium hydrogen phosphate dodecahydrate (Wako Pure ChemicalIndustries, Ltd., Japan, MW: 358.14), and 8.77 g of sodium chloride(Junsei Chemical Co., Ltd., MW: 58.44) dissolved in Milli-Q water.

The pretreated culture supernatant (1 liter) was applied to a protein Gcolumn (Hi Trap Protein G HP, 5 ml, GE Healthcare Bio-Sciences Corp.,Japan) equilibrated with PBS (Dulecco's phosphate buffered saline,SIGMA). Thereafter, the column was washed with 25 ml or more PBS, thenwith 25 ml or more buffer prepared by adding NaCl to PBS to bring theNaCl concentration to 1.85 M, and with 30 ml of PBS again. After thecompletion of the washing procedure, 25 ml of acidic buffer was added tothe column, and the target protein was recovered. The target protein wasneutralized with a neutralizing buffer immediately after it wasrecovered. AKTAexplorer 10s (GE Healthcare Bio-Sciences Corp, Japan) wasused in the separation and purification procedure. Endotoxin was removedbefore use.

11-3-3. Preparation of Purified Authentic Sample

The purified authentic sample obtained in Example 11-3-2 wasconcentrated using an ultrafilter membrane VIVASPIN20 10,000 MWCO PES(Sartorius Stedim Japan K. K., Japan). Thereafter, the buffer in thesample was substituted with PBS using NAP-25 Columns (GE HealthcareBio-Sciences Corp, Japan). After the completion of the concentration andsubstitution procedures, the resultanting solution was filtered througha 0.22 μm filter (Millex G V; Millipore, Japan).

A protein concentration was determined by measuring a specificabsorbance at 280 nm (A280 nm) (E1%, 1 cm=10.6).

Example 12

Analysis of Mouse to which Recombinant mFZD1crd-hFcm has beenAdministered

12-1. Administration to Mouse

In accordance with the method described in Example 8-1, the recombinantmFZD1crd-hFcm obtained in Example 11-3 was administered to mice in orderto evaluate physiological effects thereof on bone tissue.

Since the recombinant mFZD1crd-hFcm is a protein comprising the humanantibody Fc region, the possibility of suppression of activity of therecombinant mFZD1crd-hFcm upon production of the neutralizing antibodyin the body resulting from administration was considered. In order toreduce a risk of production of the neutralizing antibody, accordingly,homozygotes (97 KD mice, CLEA Japan, Inc., Proc. Natl. Acad. Sci.,U.S.A., 97: 722-7, 2000) obtained via back-crossing of theimmunoglobulin μ chain gene knockout mice lacking functional Blymphocytes and producing no antibodies into the MCH (ICR) strain (CLEAJapan, Inc.) were used. Mice were divided into groups based on bodyweights at age of 5 weeks on the previous day of the initiation ofadministration (i.e., day-1).

The recombinant mFZD1crd-hFcm was diluted with PBS to adjust a proteinconcentration to 5 mg/ml, and then administered into the tail veins ofmice of the recombinant mFZD1crd-hFcm test group in amounts of 200 μlper mouse once every 10 days (seven times in total). As a control groupfor comparison of changes in bone tissue, a non-treatment group wasdesignated. The day of the initial administration was designated as day0, the recombinant was administered to the tail vein every 10 days up today 60 (seven times in total), and mice were subjected to necropsy onday 70.

12-2. Necropsy Finding

The femurs, sternums, and craniums of 5 mice to which the recombinantmFZD1crd-hFcm had been administered were observed at necropsy. As aresult, whitening and epiphyseal hypertrophy of the femur, whitening ofthe sternum, whitening and hardening of the cranium, and hardening ofthe costa were observed as characteristic changes in mice to which therecombinant mFZD1crd-hFcm had been administered compared with thecontrol mice. The number of mice exhibiting changes is described below.

12-2-1. Femur

At necropsy, whitening was observed in 4 mice, a certain degree ofwhitening was observed in a mouse, epiphyseal hypertrophy was observedin another mouse among the 5 mice to which the mFZD1crd-hFcmrecombinants had been administered, and a certain degree of whiteningwas observed in a mouse among 5 control mice.

12-2-2. Sternum

At necropsy, whitening was observed in 4 mice and a certain degree ofwhitening was observed in a mouse among the 5 mice to which themFZD1crd-hFcm recombinants had been administered, and a certain degreeof whitening was observed in a mouse among 5 control mice.

12-2-3. Cranium

At necropsy, whitening and hardening were observed in 4 mice and acertain degree of whitening and hardening was observed in a mouse amongthe 5 mice to which the mFZD1crd-hFcm recombinants had beenadministered, and no change was observed in all 5 control mice.

12-2-4. Costa

At necropsy, hardening was observed in a mouse among the 5 mice to whichthe recombinant mFZD1crd-hFcm had been administered, although no changewas observed in all 5 control mice.

The above results demonstrate the possibility that whitening andepiphyseal hypertrophy of the femur, whitening of the sternum, whiteningand hardening of the cranium, and hardening of the costa were induced byadministration of the recombinant mFZD1crd-hFcm.

Example 13 13-1. Confirmation of Expression of the Fusion Protein ofMouse FZD7 Extracellular Cysteine-Rich Domain and Human Fc Mutant in 4-,8-, and 16-Week-Old USmFZD7crd-hFcm KI Chimeric Mice

The concentrations of the fusions of the mouse FZD7 extracellularcysteine-rich domains and the human Fc mutants in the serum samples ofthe 4-week-old USmFZD7crd-hFcm KI chimeric mice (6 female mice), the4-week-old control mice (6 female mice), the 8-week-old USmFZD7crd-hFcmKI chimeric mice (6 female mice), the 8-week-old control mice (6 femalemice), the 16-week-old USmFZD7crd-hFcm KI chimeric mice (6 female miceand 6 male mice), and the 16-week-old control mice (6 female mice and 5male mice) prepared in accordance with the method described in Example 1were detected via ELISA by the method described in Example 2. Mice wereraised while humidity, temperature, and light conditions were keptconstant (temperature: 22° C.; humidity: 55%; and 12 hours light and 12hours darkness) where they were allowed to freely eat feeds (CE-2, CLEAJapan, Inc.).

As a result, the average concentration among the 4-week-old femaleUSmFZD7crd-hFcm KI chimeric mice was found to be 61.2 μg/ml, that amongthe 8-week-old female mice was found to be 220.4 μg/ml, that among the16-week-old female mice was found to be 277.4 μg/ml, that among the16-week-old male mice was found to be 253.3 μg/ml, and theconcentrations in all control mice were lower than the detection limit.

The above results suggest that the fusion of the mouse FZD7extracellular cysteine-rich domain and the human Fc mutant is expressedin the bodies of 4-week or older mice and circulated in the blood. Theresults also suggest that the concentration of the fusion is elevatedwith age.

13-2. Tibial Bone Morphometry Using 4-, 8-, and 16-Week-OldUSmFZD7crd-hFcm KI Chimeric Mice 13-2-1. Bone Morphometry

In order to obtain the data regarding the mineral apposition rate, themineralization surface, and the bone formation rate, calcein (ProductNumber: 340-00433, Dojindo Laboratories, Japan) was dissolved in 2%sodium bicarbonate solution (Product Number: 37116-00, Kanto ChemicalCo., Inc., Japan), and the prepared calcein solution (a calciumchelator) was administered subcutaneously at a dose of 16 mg/kg prior tonecropsy. In the case of necropsy at age of 4 weeks, calcein wasadministered 3 days and 1 day before necropsy. In the case of necropsyat age of 8 weeks and 16 weeks, calcein was administered 6 days and 1day before necropsy. Tibiae were sampled from 4-, 8-, and 16-week-oldmice at necropsy, samples of undemineralized tibial sections wereprepared, and the samples were then subjected to toluidine blue staining(TB staining), alkaline phosphatase staining (ALP staining), andtartrate-resistant acid phosphatase staining (TRAP staining). In orderto prepare section samples, the tibia samples were embedded in GMA(glycolmethacrylate) resin in advance. The metaphyseal secondarycancellous bones of the obtained samples of undemineralized sectionswere subjected to measurement of the bone volume/tissue volume as thebone structure parameter (BV/TV), the osteoblast number/bone perimeteras the bone formation parameter (Ob.N/B.Pm), the osteoblast surface/bonesurface (Ob.S/BS), the osteoid volume/bone volume (OV/BV), the mineralapposition rate (MAR), the mineralization surface/bone surface (MS/BS),the bone formation rate/bone surface (BFR/BS), the osteoclastnumber/bone perimeter as the bone absorption parameter (Oc.N/B.Pm), andthe osteoclast surface/bone surface (Oc.S/BS).

13-2-2. Bone Volume/Tissue Volume

As a result of the measurement of the bone volume/tissue volume (BV/TV)of tibia samples obtained from 6 female control mice and 6 femaleUSmFZD7crd-hFcm KI chimeric mice subjected to necropsy at ages of 4, 8,and 16 weeks, increases were observed in the bone volume/tissue volumeof the group of USmFZD7crd-hFcm KI chimeric mice at ages of 4, 8, and 16weeks compared with the control group. This suggests the possibilitythat the increased bone volume/tissue volume in the secondary cancellousbone of the tibial metaphysis was induced by overexpression of the mouseFZD7 extracellular cysteine-rich domain-human Fc mutant fusionconstructs.

Further, tibia samples obtained from 5 male control mice and 6 maleUSmFZD7crd-hFcm KI chimeric mice subjected to necropsy at age of 16weeks were subjected to measurement of the bone volume/tissue volume. Asa result, the bone volume/tissue volume of the group of USmFZD7crd-hFcmKI chimeric mice was found to have been increased compared with that ofthe control group (with a significant difference). The resultsdemonstrate the possibility that increased bone volume/tissue volume inthe secondary cancellous bone of the tibial metaphyseal end was causedby overexpression of the fusion protein of mouse FZD7 extracellularcysteine-rich domain and human Fc mutant in male mice as well as infemale mice (Table 5).

TABLE 5 Bone volume/ tissue volume (BV/TV) Age/sex/transgene Average  4W ♀ USmFZD7crd-hFcm KI/Control 11.8/7.4  8 W ♀ USmFZD7crd-hFcmKI/Control 16.1/5.2 16 W ♀ USmFZD7crd-hFcm KI/Control 18.5/4.5 16 W ♂USmFZD7crd-hFcm KI/Control 15.4/5.3

13-2-3. Osteoblast Number/Bone Perimeter, Osteoblast Surface/BoneSurface, and Osteoid Volume/Bone Volume

The tibia samples obtained from 6 female control mice and 6 femaleUSmFZD7crd-hFcm KI chimeric mice subjected to necropsy at ages of 4, 8,and 16 weeks were subjected to measurement of the osteoblast number/boneperimeter, the osteoblast surface/bone surface, and the osteoidvolume/bone volume. As a result, the osteoblast number/bone perimeterand the osteoid volume/bone volume were found to be likely to decreasein the group of USmFZD7crd-hFcm KI chimeric mice at ages of 4 weeks and8 weeks compared with the control group. This demonstrates that theosteoblast number/bone perimeter and the osteoid volume/bone volume inthe secondary cancellous bone of the tibial metaphyseal end may not besubstantially influenced or somewhat suppressed by overexpression of thefusion protein of mouse FZD7 extracellular cysteine-rich domain andhuman Fc mutant at young age. There was substantially no difference interms of the osteoblast surface/bone surface between the recombinantmice and the control mice at ages of 4, 8, and 16 weeks.

As a result of measurement of the osteoblast number/bone perimeter, theosteoblast surface/bone surface, and the osteoid volume/bone volume ofthe tibia samples obtained from 5 male control mice and 6 maleUSmFZD7crd-hFcm KI chimeric mice subjected to necropsy at age of 16weeks, substantially no difference was observed therebetween. Thisindicates that male mice are not substantially influenced byoverexpression of the fusion protein of mouse FZD7 extracellularcysteine-rich domain and human Fc mutant fusion constructs as with thecase of female mice (Table 6).

TABLE 6 Osteoblast Osteoblast surface/ number/bone bone perimetersurface Osteoidvolume/ (Ob. N/ (Ob. S/ bone volume B. Pm) BS) (OV/BV)Age/sex/transgene Average Average Average 4 W ♀ USmFZD7crd-hFcm2219/2710 28.3/29.8 2.2/3   KI/Control 8 W ♀ USmFZD7crd-hFcm 1387/196320.2/23.9 1.1/2.3 KI/Control 16 W ♀ USmFZD7crd-hFcm 1255/1197 15.9/15.21.42/1.28 KI/Control 16 W ♂ USmFZD7crd-hFcm 733/626 9.7/7.5 0.26/0.38KI/Control

13-2-4. Mineral Apposition Rate, Mineralization Surface, and BoneFormation Rate

As a result of measurement of the mineral apposition rate, themineralization surface, and the bone formation rate of the tibia samplesobtained from 6 female control mice and 6 female USmFZD7crd-hFcm KIchimeric mice subjected to necropsy at ages of 4, 8, and 16 weeks,increase was observed in the mineral apposition rate only at age of 16weeks, increase was observed in the mineralization surface/bone surfaceat ages of 4 and 8 weeks, and increase was observed in the boneformation rate at ages of 4, 8, and 16 weeks in the group ofUSmFZD7crd-hFcm KI chimeric mice compared with the control group. Thisindicates that mineralization of the secondary cancellous bone of thetibial metaphyseal end may have been accelerated by overexpression ofthe fusion protein of mouse FZD7 extracellular cysteine-rich domain andhuman Fc mutant.

As a result of measurement of the mineral apposition rate, themineralization surface, and the bone formation rate of the tibia samplesobtained from 5 male control mice and 6 male USmFZD7crd-hFcm KI chimericmice subjected to necropsy at age of 16 weeks, further, increases wereobserved in the chimeric mice. This indicates that mineralization of thesecondary cancellous bone of the tibial metaphyseal end may have beenaccelerated by overexpression of the fusion protein of mouse FZD7extracellular cysteine-rich domain and human Fc mutant as with the caseof femal mice (Table 7).

TABLE 7 Bone Mineral Mineralization formation apposition surface/bonerate/bone rate surface surface (MAR) (MS/BS) (BFR/BS) Age/sex/transgeneAverage Average Average 4 W ♀ USmFZD7crd-hFcm 3.9/3.9 25.3/18.436.6/26.4 KI/Control 8 W ♀ USmFZD7crd-hFcm 2.5/2.5 19.5/15   18.3/14.2KI/Control 16 W ♀ USmFZD7crd-hFcm 1.6/1.2 18.7/17.1 11.4/7.9  KI/Control16 W ♂ USmFZD7crd-hFcm 1.2/0.9 17.7/11.3 7.9/3.8 KI/Control

13-2-5. Osteoclast Number/Bone Perimeter and Osteoclast Surface/BoneSurface

As a result of measurement of the osteoclast number/bone perimeter andthe osteoclast surface/bone surface of the tibia samples obtained from 6female control mice and 6 female USmFZD7crd-hFcm KI chimeric micesubjected to necropsy at age of 4, 8, and 16 weeks, no difference wasobserved therebetween. This indicates that the osteoclast number/boneperimeter and the osteoclast surface/bone surface of the secondarycancellous bone of the tibial metaphyseal end may not be substantiallyinfluenced by overexpression of the fusion protein of mouse FZD7extracellular cysteine-rich domain and human Fc mutant.

As a result of measurement of the osteoclast number/bone perimeter andthe osteoclast surface/bone surface of the tibia samples obtained from 5male control mice and 6 male USmFZD7crd-hFcm KI chimeric mice subjectedto necropsy at age of 16 weeks, further, no difference was observedtherebetween. This indicates that the osteoclast number/bone perimeterand the osteoclast surface/bone surface of the secondary cancellous boneof the tibial metaphyseal end may not be influenced by overexpression ofthe fusion protein of mouse FZD7 extracellular cysteine-rich domain andhuman Fc mutant as with the case of female mice (Table 8).

TABLE 8 Osteoclast number/ Osteoclast surface/ bone perimeter bone (Oc.N/B. Pm) surface (OC. S/BS) Age/sex/transgene Average Average 4 W ♀USmFZD7crd-hFcm 380.8/381.8 4.2/6.2 KI/Control 8 W ♀ USmFZD7crd-hFcm265.7/289.9   4/4.6 KI/Control 16 W ♀ USmFZD7crd-hFcm 163.7/181.82.2/1.9 KI/Control 16 W ♂ USmFZD7crd-hFcm 115.5/112.9 1.5/1.4 KI/Control

13-3. Measurement of Bone Strength

The femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom 6 female control mice and 6 female USmFZD7crd-hFcm KI chimeric micesubjected to necropsy at ages of 4, 8, and 16 weeks, the measured valueswere found to have increased at both ages of 8 weeks and 16 weeks in thegroup of USmFZD7crd-hFcm KI chimeric mice compared with the controlgroup. This indicates that the increased maximum load of the femur mayhave been caused by overexpression of the fusion protein of mouse FZD7extracellular cysteine-rich domain and human Fc mutant.

As a result of measurement of the maximum load of femur samples obtainedfrom 5 male control mice and 6 male USmFZD7crd-hFcm KI chimeric micesubjected to necropsy at age of 16 weeks, further, the values were foundto have increased in the group of USmFZD7crd-hFcm KI chimeric micecompared with the control group. This indicates that an increase in themaximum load of the femur may have been induced by overexpression of thefusion protein of mouse FZD7 extracellular cysteine-rich domain andhuman Fc mutant as with the case of female mice (Table 9).

TABLE 9 Maximum load (N) Age/sex/transgene Average  4 W ♀USmFZD7crd-hFcm KI/Control 11.1/11.3  8 W ♀ USmFZD7crd-hFcm KI/Control23.3/17.4 16 W ♀ USmFZD7crd-hFcm KI/Control 35.3/26.6 16 W ♂USmFZD7crd-hFcm KI/Control 35.2/22.8

13-4. Analysis of Bone Structure (3-Dimensional Microfocus X-Ray CT)

The left tibia samples were obtained at necropsy, and the internalstructure of the cancellous bone region of the proximal tibialmetaphysis was observed using a high-resolution microfocus X-ray CTscanner (micro-CT, Scan Xmate-L090, Comscantecno Co., Ltd., Japan) andthe analytic software (TRY 3D-BON, Ratoc System Engineering Co., Ltd.,Japan) in a non-invasive manner. The bone volume/tissue volume (BV/TV),the trabecular thickness (Tb. Th), the trabecular number (Tb. N), thetrabecular separation (Tb. Sp), and the trabecular spacing (Tb. Spac)were measured.

The internal structure of the cancellous bone of the femur samplesobtained from 6 female control mice and 6 female USmFZD7crd-hFcm KIchimeric mice subjected to necropsy at ages of 4, 8, and 16 weeks wasobserved via micro CT. As a result, the average bone volume/tissuevolume, trabecular thickness, and trabecular number were found to haveincreased, and the average trabecular separation and trabecular spacingwere found to have decreased in the group of USmFZD7crd-hFcm KI chimericmice compared with the control group. In addition, the results obtainedvia micro CT using the femur samples obtained from 6 male control miceand 6 male USmFZD7crd-hFcm KI chimeric mice subjected to necropsy at ageof 16 weeks were similar to those obtained from female mice at ages 4,8, and 16 weeks. This suggests that the increased bone volume/tissuevolume, the increased trabecular thickness, the increased trabecularnumber, the decreased trabecular separation, and the decreasedtrabecular spacing in the cancellous bone of the proximal tibialmetaphysis may have been induced by overexpression of the fusion proteinof mouse FZD7 extracellular cysteine-rich domain and human Fc mutant(Table 10).

TABLE 10 Bone Trabecular Trabecular Trabecular volume/tissue thicknessnumber Trabecular spacing volume (Tb. Th, (Tb. N, separation (Tb. Spac,(BV/TV, %) μm) 1/mm) (Tb. Sp, μm) μm) Age/sex/transgene Average AverageAverage Average Average 4 W ♀   22.9/12.3 32.1/27.5   7/4.3 111.2/216.8143.3/244.3 USmFZD7crd-hFcm KI/4 W control 8 W ♀   21.2/6.8 40.8/28.85.1/2.2 157.6/436.8 198.4/465.6 USmFZD7crd-hFcm KI/8 W control 16 W ♀22.1/5 51.2/32.5 4.2/1.5 190.6/797.4 241.9/830   USmFZD7crd-hFcm KI/16 Wcontrol 16 W ♂ 17.5/6 41.2/29.4 4.1/2     203/510.6 244.3/540  USmFZD7crd-hFcm KI/16 W control

Example 14 14-1. Confirmation of Expression of the Fusion of Human FZD7Extracellular Cysteine-Rich Domain and Human Fc Mutant in 8- and12-Week-Old UShFZD7crd-hFcm KI Chimeric Mice

The fusion of human FZD7 extracellular cysteine-rich domain and human Fcmutant existing in the serum samples of the 8-week-old UShFZD7crd-hFcmKI chimeric mice (6 female mice), the 8-week-old control mice (6 femalemice), the 8-week-old UShFZD7crd-hFcm KI chimeric mice (6 male mice),the 8-week-old control mice (6 male mice), the 12-week-oldUShFZD7crd-hFcm KI chimeric mice (6 male mice), and the 12-week-oldcontrol mice (6 male mice) prepared in accordance with the methoddescribed in Example 3 were detected via ELISA in accordance with themethod described in Example 2. Mice were raised while humidity,temperature, and light conditions were kept constant (temperature: 22°C.; humidity: 55%; and 12 hours light and 12 hours darkness) where theywere allowed to freely eat feeds (CE-2, CLEA Japan, Inc.).

As a result, the average concentration among the 8-week-old female micewas found to be 244.0 μg/ml, that among the 8-week-old male mice wasfound to be 190.2 μg/ml, that among the 12-week-old male mice was foundto be 208.1 μg/ml, and the concentrations assayed with the use of theserum samples obtained from control mice were lower than the detectionlimit.

The above results suggest that the fusion protein of the human FZD7extracellular cysteine-rich domain and the human Fc mutant are expressedin the bodies of 8-week-old or older mice and circulated in the blood.

14-2. Necropsy Finding of 8-Week-Old UShFZD7crd-hFcm KI Chimeric Mice

The chimeric mice prepared in Example 3 (6 female mice and 6 male mice)were subjected to necropsy at age of 8 weeks, and the spleen, the femur,the sternum, the cranium, the spondylus, and the costa were observed. Asa result, whitening of the femur, whitening of the sternum, whiteningand hardening of the cranium, and a certain degree of hardening of thecosta were observed as characteristic changes in the UShFZD7crd-hFcm KIchimeric mice compared with the control mice (6 female mice and 6 malemice). In addition, spleen enlargement was observed in theUShFZD7crd-hFcm KI chimeric mice. The number of mice exhibiting changesis described below.

14-2-1. Necropsy Finding of Femur

Whitening was observed in 10 mice and a certain degree of whitening wasobserved in 2 mice among the 12 UShFZD7crd-hFcm KI chimeric micesubjected to necropsy compared with 12 control mice.

14-2-2. Necropsy Finding of Sternum

Whitening was observed in 10 mice and a certain degree of whitening wasobserved in 2 mice among the 12 UShFZD7crd-hFcm KI chimeric micesubjected to necropsy compared with 12 control mice.

14-2-3. Necropsy Finding of Cranium

Whitening was observed in 10 mice, a certain degree of whitening wasobserved in 2 mice, hardening was observed in a mouse, and a certaindegree of hardening was observed in 9 mice among the 12 UShFZD7crd-hFcmKI chimeric mice subjected to necropsy compared with 12 control mice.

14-2-4. Necropsy Finding of Costa

A certain degree of hardening was observed in 5 mice among the 12UShFZD7crd-hFcm KI chimeric mice subjected to necropsy compared with 12control mice.

14-2-5. Necropsy Finding of Spleen

Tendency of spleen enlargement was observed in 6 mice among the 12UShFZD7crd-hFcm KI chimeric mice subjected to necropsy compared with 12control mice.

The above results indicate that whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, and a certain degree ofhardening of the costa may have been induced by overexpression of thehuman FZD7 extracellular cysteine-rich domain-human Fc mutant fusionconstructs.

14-3. Measurement of Bone Strength of 8-Week-Old UShFZD7crd-hFcm KIChimeric Mice

The femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom 12 control mice and 12 UShFZD7crd-hFcm KI chimeric mice, themeasured values were found to have increased in both female and malemice in the group of USmFZD7crd-hFcm KI chimeric mice compared with thecontrol group. This indicates that the increased maximum load of thefemur may have been induced by overexpression of the fusion protein ofhuman FZD7 extracellular cysteine-rich domain and human Fc mutant (Table11).

TABLE 11 Maximum load (N) Age/sex/transgene Average 8 W ♀UShFZD7crd-hFcm KI/Control 22.5/19.3 8 W ♂ UShFZD7crd-hFcm KI/Control24.3/19.8

14-4. Analysis of Bone Structure of 8-Week-Old UShFZD7crd-hFcm KIChimeric Mouse (3-Dimensional Microfocus X-Ray CT)

The femur samples were obtained at necropsy, and the internal structureof the cancellous bone region of the distal femoral metaphysis wasobserved using a high-resolution microfocus X-ray CT scanner (micro-CT,Scan Xmate-L090, Comscantecno Co., Ltd.) and the analytic software (TRY3D-BON, Ratoc System Engineering Co., Ltd.) in a non-invasive manner.The bone volume/tissue volume (BV/TV), the trabecular thickness (Tb.Th), the trabecular number (Tb. N), the trabecular separation (Tb. Sp),and the trabecular spacing (Tb. Spac) were measured.

As a result of observation of the internal structure of the cancellousbone region of the femur samples obtained from control mice (6 femalemice and 6 male mice) and the UShFZD7crd-hFcm KI chimeric mice (6 femalemice and 6 male mice) via micro-CT, the average bone volume/tissuevolume, trabecular thickness, and trabecular number were found to haveincreased, and the average trabecular separation and trabecular spacingwere found to have decreased in the group of UShFZD7crd-hFcm KI chimericmice compared with the control group. It was thus suggested that theincreased bone volume/tissue volume, the increased trabecular thickness,the increased trabecular number, the decreased trabecular separation,and the decreased trabecular spacing in the cancellous bone of thedistal femoral metaphysis may have been induced by overexpression of thefusion protein of human FZD7 extracellular cysteine-rich domain andhuman Fc mutant (Table 12).

TABLE 12 Average bone Trabecular Trabecular Trabecular volume/tissuethickness number Trabecular spacing volume (Tb. Th, (Tb. N, separation(Tb. Spac, (BV/TV, %) μm) 1/mm) (Tb. Sp, μm) μm) Age/sex/transgeneAverage Average Average Average Average 8 W ♀ UShFZD7crd-hFcm 25.1/7.643.1/28.2 5.7/2.6  130.8/367.5   174/395.7 KI/8 W control 8 W ♂UShFZD7crd-hFcm 16.3/8.6 34.1/28.4 4.7/3.02 178.6/312.1 212.8/340.6 KI/8W control

14-5. Necropsy Finding of 12-Week-Old UShFZD7crd-hFcm KI Chimeric Mouse

The chimeric mice prepared in Example 3 were subjected to necropsy (6male mice) at age of 12 weeks, and the spleen, the femur, the sternum,the cranium, the spondylus, and the costa were observed. As a result,whitening of the femur, whitening of the sternum, whitening andhardening of the cranium, and a certain degree of hardening of thespondylus were observed as characteristic changes in the UShFZD7crd-hFcmKI chimeric mice compared with the control mice (6 male mice). Inaddition, a certain degree of tendency of spleen enlargement wasobserved in the UShFZD7crd-hFcm KI chimeric mice. The number of miceexhibiting changes is described below.

14-5-1. Necropsy Finding of Femur

Whitening was observed in a mouse and a certain degree of whitening wasobserved in 4 mice among the 6 UShFZD7crd-hFcm KI chimeric micesubjected to necropsy compared with 6 control mice.

14-5-2. Necropsy Finding of Sternum

Whitening was observed in a mouse and a certain degree of whitening wasobserved in a mouse among the 6 UShFZD7crd-hFcm KI chimeric micesubjected to necropsy compared with 6 control mice.

14-5-3. Necropsy Finding of Cranium

Whitening was observed in 2 mice, a certain degree of whitening wasobserved in 3 mice, hardening was observed in a mouse, and a certaindegree of hardening was observed in a mouse among the 6 UShFZD7crd-hFcmKI chimeric mice subjected to necropsy compared with 6 control mice.

14-5-4. Necropsy Finding of Spondylus

A certain degree of hardening was observed in a mouse among the 6UShFZD7crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice.

14-5-5. Necropsy Finding of Spleen

Spleen enlargement was observed in a mouse among the 6 UShFZD7crd-hFcmKI chimeric mice subjected to necropsy compared with 6 control mice.

The above results indicate that whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, and a certain degree ofhardening of the spondylus may have been induced by overexpression ofthe fusion protein of human FZD7 extracellular cysteine-rich domain andhuman Fc mutant.

14-6. Pathological Finding of 12-Week-Old UShFZD7crd-hFcm KI ChimericMouse

The H&E stained femur and sternum pathological sections obtained fromsix 12-week-old control chimeric mice and six UShFZD7crd-hFcm KIchimeric mice were observed. As a result, the thickened femoraldiaphyseal wall (FIGS. 14 and 15, Table 13), the increased cancellousbone (FIG. 16), and the increased sternal cancellous bone (FIG. 17) wereobserved in the UShFZD7crd-hFcm KI chimeric mice compared with controlmice. The number of mice exhibiting changes is described below.

14-6-1. Femur

The increased cancellous bone was observed in all the 6 UShFZD7crd-hFcmKI chimeric mice subjected to necropsy compared with 6 control mice(FIG. 16). Further, transected sections obtained from 3 femoral sites(i.e., sites 30%, 50%, and 80% away from the proximal end) weresubjected to measurement of the diaphyseal wall thickness. As a result,the average minimum wall thickness at a site 30% away from the proximalend and the average maximum wall thickness at a site 50% away from theproximal end were found to be larger than those of the control group(FIGS. 14 and 15, Table 13).

TABLE 13 Minimum diaphyseal Maximum diaphyseal wall thickness at sitewall thickness at site 30% away from 50% away from proximal end (mm)proximal end (mm) Age/sex/transgene Average Average 12 W ♂ UShFZD7crd-0.22/0.19 0.47/0.34 hFcm KI/12 W control

14-6-2. Sternum

The increased cancellous bone was observed in 5 mice among the 6UShFZD7crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice (FIG. 17).

The above results demonstrate that the thickened femoral diaphysealwall, the increased cancellous bone, and the increased sternalcancellous bone may have been caused by overexpression of the fusionprotein of human FZD7 extracellular cysteine-rich domain and human Fcmutant.

14-7. Biochemical Analysis of Serum

Six 12-week-old UShFZD7crd-hFcm KI male chimeric mice and 6 male controlmice were exsanguinated under ether anesthesia to prepare serum samples.With the use of Hitachi 7180 (Hitachi Science Systems Ltd., Japan),serum samples were subjected to biochemical analysis (LDH activity, GOTactivity, GPT activity, CK activity, ALP activity, AMY activity, LAPactivity, LIP activity, T-CHO concentration, F-CHO concentration,LDL-CHO concentration, HDL-CHO concentration, TG concentration, PLconcentration, GLU concentration, GA %, UA concentration, BUNconcentration, CREA concentration, T-BIL concentration, D-BILconcentration, TP concentration, ALB concentration, A/G ratio, IPconcentration, Ca concentration, Mg concentration, Na concentration, Kconcentration, Cl concentration, Fe concentration, UIBC concentration,and TIBC concentration). As a result, the values obtained with the useof UShFZD7crd-hFcm KI chimeric mice were not significantly differentfrom those of the control mice.

Example 15 15-1. Preparation of Ovariectomized (OVX) Mouse Models

In order to evaluate the efficacy of the recombinant mFZD7crd-hFcm as atherapeutic agent for osteoporosis, ovariectomized (OVX) mouse modelswere prepared. Since the recombinant mFZD7crd-hFcm is a proteincomprising the human antibody Fc region, the possibility of suppressingthe activity of recombinant mFZD7crd-hFcm upon production of theneutralizing antibody in the body resulting from administration wasconsidered. In order to reduce a risk of production of the neutralizingantibody, accordingly, homozygotes (97 KD mice, CLEA Japan, Inc., Proc.Natl. Acad. Sci., U.S.A., 97: 722-7, 2000) obtained via back-crossing ofthe immunoglobulinμ chain gene knockout mice lacking functional Blymphocytes and producing no antibodies into the MCH (ICR) strain (CLEAJapan, Inc.) were used for preparation of OVX mouse models. The dorsalregions of 10-week-old 97 KD mice were incised under anesthesia toremove both ovaries, or the mice were subjected to a sham operation ofincision only without ovariectomy, followed by suturing.

15-2. Analysis of OVX Mouse Models to which Recombinant mFZD7crd-hFcmhas been Administered

15-2-1. Administration to OVX Mouse Models

The recombinant mFZD7crd-hFcm was administered to the OVX mouse modelsprepared in Example 15-1 one week after the surgical operation in orderto evaluate the efficacy of the recombinant mFZD7crd-hFcm in treatmentof osteoporosis. As a test material, a bisphosphonate preparation(risedronate, Wako Pure Chemical Industries, Ltd., Product Number:572-27451) was used for comparison, in addition to the recombinantmFZD7crd-hFcm, and a group to which both the recombinant mFZD7crd-hFcmand risedronate would be administered was further designated. The day atwhich administration was initiated was designated as day 0, and necropsywas carried out at day 69 and day 70. The recombinant mFZD7crd-hFcm wasadministered into the caudal veins (IV) in amounts of 1 mg/dose onceevery 10 days (seven times in total). Risedronate was administeredsubcutaneously (SC) in amounts of 5 μg/kg/dose three times in a week (30times in total). Groups were designated as follows: a group subjected tosham operation without administration of a test substance (i.e., thesham/non-treatment group); a group subjected to OVX and administrationof risedronate (i.e., the OVX/risedronate group); a group subjected tosham operation and administration of risedronate (i.e., thesham/risedronate group); a group subjected to OVX and administration ofthe recombinant mFZD7crd-hFcm (i.e., the OVX/mFZD7crd-hFcm group); agroup subjected to sham operation and administration of the recombinantmFZD7crd-hFcm (i.e., the sham/mFZD7crd-hFcm group); a group subjected toOVX and administration of the recombinant mFZD7crd-hFcm and risedronate(i.e., the OVX/mFZD7crd-hFcm/risedronate group); and a group subjectedto OVX without administration of a test substance (i.e., theOVX/non-treatment group). These groups were subjected to theadministration experiment.

15-2-2. Necropsy Finding of OVX Mouse Models to which the RecombinantmFZD7crd-hFcm had been Administered

The mice described in Example 15-2 were subjected to necropsy on day 69and day 70, the femur, the sternum, the cranium, the costa, thespondylus, the spleen, and the uterus were observed. As a result, thesham/risedronate group, the OVX/mFZD7crd-hFcm group, thesham/mFZD7crd-hFcm group, and the OVX/mFZD7crd-hFcm/risedronate groupexhibited characteristic changes in terms of whitening of the femur,whitening of the sternum, whitening and hardening of the cranium,hardening of the costa, and hardening of the spondylus (except for thesham/risedronate group) compared with the sham/non-treatment group. Thenumber of mice exhibiting a certain degree of blackening in the spleenincreased in the sham/mFZD7crd-hFcm group. Uterine involution wasobserved in the OVX/risedronate group, the OVX/mFZD7crd-hFcm group, theOVX/mFZD7crd-hFcm/risedronate group, and the OVX/non-treatment group.The number of mice exhibiting changes in the aforementioned organs isdescribed below.

15-2-2-1. Necropsy Finding of Femur

While a certain degree of whitening was observed in 1 mouse among 10mice of the sham/non-treatment group, whitening was observed in 1 mouseamong 10 mice of the OVX/risedronate group, whitening was observed in 2mice and a certain degree of whitening was observed in 4 mice among 10mice of the sham/risedronate group, whitening was observed in 5 mice anda certain degree of whitening was observed in 4 mice among 10 mice ofthe OVX/mFZD7crd-hFcm group, whitening was observed in 8 mice and acertain degree of whitening was observed in 2 mice among 10 mice of thesham/mFZD7crd-hFcm group, whitening was observed in 7 mice and a certaindegree of whitening was observed in 3 mice among 10 mice of theOVX/mFZD7crd-hFcm/risedronate group, and a certain degree of whiteningwas observed in a mouse among 10 mice of the OVX/non-treatment group.

15-2-2-2. Necropsy Finding of Sternum

While a certain degree of whitening was observed in 1 mouse among 10mice of the sham/non-treatment group, a certain degree of whitening wasobserved in 2 mice among 10 mice of the OVX/risedronate group, whiteningwas observed in 4 mice and a certain degree of whitening was observed in3 mice among 10 mice of the sham/risedronate group, whitening wasobserved in 4 mice and a certain degree of whitening was observed in 3mice among 10 mice of the OVX/mFZD7crd-hFcm group, whitening wasobserved in 8 mice and a certain degree of whitening was observed in amouse among 10 mice of the sham/mFZD7crd-hFcm group, whitening wasobserved in 8 mice and a certain degree of whitening was observed in 1mouse among 10 mice of the OVX/mFZD7crd-hFcm/risedronate group, and acertain degree of whitening was observed in a mouse and deepening incolor was observed in a mouse among 10 mice of the OVX/non-treatmentgroup.

15-2-2-3. Necropsy Finding of Cranium

While a certain degree of whitening was observed in 1 mouse among 10mice of the sham/non-treatment group, a certain degree of whitening wasobserved in 3 mice and a certain degree of hardening was observed in 1mouse among 10 mice of the OVX/risedronate group, whitening was observedin 2 mice, a certain degree of whitening was observed in 6 mice, and acertain degree of hardening was observed in 2 mice among 10 mice of thesham/risedronate group, whitening was observed in 4 mice, a certaindegree of whitening was observed in 3 mice, hardening was observed in 2mice, and a certain degree of hardening was observed in 3 mice among 10mice of the OVX/mFZD7crd-hFcm group, whitening was observed in 7 mice, acertain degree of whitening was observed in a mouse, hardening wasobserved in 5 mice, and a certain degree of hardening was observed in 4mice among 10 mice of the sham/mFZD7crd-hFcm group, whitening wasobserved in 5 mice, a certain degree of whitening was observed in 2mice, hardening was observed in 2 mice, and a certain degree ofhardening was observed in 4 mice among 10 mice of theOVX/mFZD7crd-hFcm/risedronate group, and a certain degree of whiteningwas observed in 1 mouse and partial softening was observed in 2 miceamong 10 mice of the OVX/non-treatment group.

15-2-2-4. Necropsy Finding of Costa

In comparison with the sham/non-treatment group (10 mice), hardening wasobserved in 1 mouse among 10 mice of the OVX/risedronate group,hardening was observed in 2 mice and a certain degree of hardening wasobserved in 1 mouse among 10 mice of the sham/risedronate group,hardening was observed in 2 mice and a certain degree of hardening wasobserved in 2 mice among 10 mice of the OVX/mFZD7crd-hFcm group,hardening was observed in 3 mice and a certain degree of hardening wasobserved in 2 mice among 10 mice of the sham/mFZD7crd-hFcm group,hardening was observed in 2 mice and a certain degree of hardening wasobserved in 2 mice among 10 mice of the OVX/mFZD7crd-hFcm/risedronategroup, and partial softening was observed in 2 mice among 10 mice of theOVX/non-treatment group.

15-2-2-5. Necropsy Finding of Spondylus

In comparison with the sham/non-treatment group (10 mice), hardening wasobserved in 1 mouse and a certain degree of hardening was observed in amouse among 10 mice of the OVX/mFZD7crd-hFcm group, hardening wasobserved in 4 mice among 10 mice of the sham/mFZD7crd-hFcm group, andhardening was observed in 2 mice and a certain degree of hardening wasobserved in 1 mouse among 10 mice of the OVX/mFZD7crd-hFcm/risedronategroup.

15-2-2-6. Necropsy Finding of Spleen

In comparison with the sham/non-treatment group (10 mice), enlargementwas observed in a mouse and blackening was observed in 1 mouse among 10mice of the OVX/risedronate group, a tendency toward enlargement wasobserved in 1 mouse and a certain degree of blackening was observed in 1mouse among 10 mice of the sham/risedronate group, a tendency towardenlargement was observed in 1 mouse and a certain degree of blackeningwas observed in 2 mice among 10 mice of the OVX/mFZD7crd-hFcm group,enlargement was observed in 1 mouse and a certain degree of blackeningwas observed in 6 mice among 10 mice of the sham/mFZD7crd-hFcm group, atendency toward enlargement was observed in 1 mouse and a certain degreeof blackening was observed in 2 mice among 9 mice of theOVX/mFZD7crd-hFcm/risedronate group, and a certain degree of blackeningwas observed in 1 mouse among 10 mice of the OVX/non-treatment group.

15-2-2-7. Necropsy Finding of Uterus

In comparison with the sham/non-treatment group (10 mice), involutionwas observed in 3 mice among 10 mice of the OVX/risedronate group,involution was observed in 4 mice and a tendency toward involution wasobserved in 2 mice among 10 mice of the OVX/mFZD7crd-hFcm group,involution was observed in 6 mice among 9 mice of theOVX/mFZD7crd-hFcm/risedronate group, and involution was observed in 3mice and a tendency toward involution was observed in 2 mice among 10mice of the OVX/non-treatment group.

The above results suggests the possibility that whitening of the femur,whitening of the sternum, whitening and hardening of the cranium, andhardening of the costa observed in the OVX/mFZD7crd-hFcm group and inthe sham/mFZD7crd-hFcm group were induced by administration of therecombinant mFZD7crd-hFcm. The above results also suggest thepossibility that whitening of the femur, whitening of the sternum,whitening and hardening of the cranium, and hardening of the costaobserved in the OVX/risedronate group and in the sham/risedronate groupwere induced by administration of risedronate. The above results furthersuggest the possibility that whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, and hardening of thecosta observed in the OVX/mFZD7crd-hFcm/risedronate group were inducedby administration of the mFZD7crd-hFcm recombinant and risedronate.Since hardening of the spondylus was observed exclusively in theOVX/mFZD7crd-hFcm group, the sham/mFZD7crd-hFcm group, and theOVX/mFZD7crd-hFcm/risedronate group, hardening of the spondylus may havebeen induced by administration of the recombinant mFZD7crd-hFcm. It wassuggested that uterine involution observed only in the OVX treatmentgroup was a change caused by OVX as reported in the literature (J. BoneMiner. Res., 20: 1085-92, 2005).

15-2-3. Pathological Finding

At necropsy conducted in Example 15-2-2, the right femur was sampledfrom each mouse, soaked and fixed in a 10% neutral buffered formalinsolution (Wako Pure Chemical Industries, Ltd., Japan), and cut intoround sections at sites 30% and 50% away from the proximal end toprepare H&E samples with lengthwise end surfaces. Changes in thecancellous bones at the ends were observed, and the maximum diaphysealwall thickness at a site 30% away from the proximal end and themaximum/minimum diaphyseal wall thickness at a site 50% away from theproximal end were measured.

In accordance with the results of observation of changes in cancellousbones at the femoral ends, evaluation was made as follows: −: no change;±; very mild; +: mild; ++; moderate; and +++; severe, and the resultsshown below were obtained.

While a very mild (±) decrease was observed in 2 mice and a mild (+)decrease was observed in 3 mice in the OVX/non-treatment group, a verymild (±) decrease was observed in 2 mice of the OVX/mFZD7crd-hFcm group.This indicates that a decrease in the cancellous bone resulting from OVXtreatment is brought back to a normal state. While a very mild (±)increase was observed in 1 mouse and a very mild (±) decrease wasobserved in 2 mice of the sham/non-treatment group, a mild (+) increasewas observed in 1 mouse of the sham/mFZD7crd-hFcm group.

As a result of the measurement of the minimum diaphyseal wall thicknessat a site 30% away from the proximal end and the maximum/minimumdiaphyseal wall thickness at a site 50% away from the proximal end, theaverage minimum diaphyseal wall thickness at a site 30% away from theproximal end and the average maximum/minimum diaphyseal wall thicknessat a site 50% away from the proximal end of the sham/mFZD7crd-hFcm groupincreased compared with the sham/non-treatment group. Compared with theOVX/non-treatment group, the average minimum diaphyseal wall thicknessat a site 30% away from the proximal end and the average maximum/minimumdiaphyseal wall thickness at a site 50% away from the proximal end ofthe OVX/mFZD7crd-hFcm group increased. When the values of theOVX/mFZD7crd-hFcm group were compared with those of thesham/non-treatment group, the average maximum diaphyseal wall thicknessat a site 50% away was equivalent to that of a group of normal controlmice (i.e., the sham/non-treatment group), and the average minimumdiaphyseal wall thickness at a site 30% away from the end and theaverage minimum diaphyseal wall thickness at a site 50% away from theend were found to be larger than those of the sham/non-treatment group(Table 14).

TABLE 14 Minimum Maximum Minimum diaphyseal diaphyseal diaphyseal wallwall wall thickness thickness thickness at site at site at site OVXnon-treatment·OVX treatment/ 30% away 50% away 50% away risedronate fromfrom from administration·recombinant proximal proximal proximaladministration·no end (mm) end (mm) end (mm) recombinant administrationAverage Average Average Sham/risedronate group vs 0.22/0.21 0.43/0.43 0.2/0.19 Sham/non-treatment group Sham/mFZD7crd-hFcm group vs 0.25/0.210.47/0.43 0.22/0.19 Sham/non-treatment group OVX/risedronate group vs0.21/0.19 0.41/0.37 0.19/0.17 OVX/non-treatment group OVX/mFZD7crd-hFcmgroup vs 0.24/0.19 0.43/0.37 0.22/0.17 OVX/non-treatment groupOVX/mFZD7crd-hFcm/risedronate 0.24/0.19 0.47/0.37 0.23/0.17 group vsOVX/non-treatment group OVX/mFZD7crd-hFcm group vs 0.24/0.21 0.43/0.430.22/0.19 Sham/non-treatment group

The above results demonstrate that the recombinant mFZD7crd-hFcm has anactivity of increasing the cancellous bone mass and an activity ofincreasing the diaphyseal wall thickness on the OVX treated mice as wellas normal control mice (i.e., Sham/non-treatment mice).

15-2-4. Measurement of Cross-Sectional Area of Femoral Cortical Bone

The right femur was sampled from each mouse at necropsy conducted inExample 15-2-2, a site 50% away from the proximal end was subjected to2D micro-CT photographing (FIG. 18), and the cross-sectional area of thecortical bone at a site 50% away from the proximal end was measured (thenumber of mice subjected to measurement: 10 mice of each group).

Compared with the sham/non-treatment group, the average of thesham/mFZD7crd-hFcm group increased. Compared with the OVX/non-treatmentgroup, the average of the OVX/mFZD7crd-hFcm group also increased. Whenthe value of the OVX/mFZD7crd-hFcm group was compared with that of thesham/non-treatment group, the average was found to be larger than thatof the group of normal control mice (i.e., the sham/non-treatmentgroup).

The above results demonstrate that the mFZD7crd-hFcm recombinant has theactivity of increasing the cross-sectional area of the cortical bone onthe OVX treated mice as well as normal control mice (i.e.,sham/non-treatment mice) (Table 15).

TABLE 15 Cross-sectional area of OVX non-treatment·OVX treatment/femoral cortical Maximum risedronate administration·recombinant bone(mm²) load (N) administration·no recombinant administration AverageAverage Sham/risedronate group vs 1.36/1.23   35/33.1 Sham/non-treatmentgroup Sham/mFZD7crd-hFcm group vs 1.43/1.23 39.2/33.1 Sham/non-treatmentgroup OVX/risedronate group vs 1.23/1.11 33.7/30.6 OVX/non-treatmentgroup OVX/mFZD7crd-hFcm group vs 1.29/1.11   36/30.6 OVX/non-treatmentgroup OVX/mFZD7crd-hFcm/risedronate group vs 1.44/1.11 38.1/30.6OVX/non-treatment group OVX/mFZD7crd-hFcm group vs 1.29/1.23   36/33.1Sham/non-treatment group

15-2-5. Measurement of Femoral Bone Strength

The right femur was sampled from each mouse at necropsy conducted inExample 15-2-2, and a three-point bending test was carried out (thenumber of mice subjected to measurement: 10 mice of each group). Whenconducting a test, the span of the support points was set as 6 mm, and aload was applied at the midpoint of the span to measure the maximum load(N).

In comparison with the sham/non-treatment group, the average of thesham/mFZD7crd-hFcm group increased. In comparison with theOVX/non-treatment group, the average of the OVX/mFZD7crd-hFcm groupincreased. When the values of the OVX/mFZD7crd-hFcm group were comparedwith those of the sham/non-treatment group, the average was found to belarger than that of the group of normal control mice (i.e., thesham/non-treatment group).

The above results demonstrate that the recombinant mFZD7crd-hFcm has theactivity of increasing the femoral bone strength on the OVX treated miceas well as normal control mice (i.e., sham/non-treatment mice) (Table15).

Example 16 16. Analysis of USmFZD1crd-hFcm KI Chimeric Mouse 16-1.Biochemical Analysis of Serum

Fourteen 16-week-old USmFZD1crd-hFcm KI female chimeric mice, 6USmFZD1crd-hFcm KI male chimeric mice, sixteen female control mice, andfourteen male control mice prepared in Example 4 were exsanguinatedunder ether anesthesia to prepare serum samples. With the use of Hitachi7180 (Hitachi Science Systems Ltd., Japan), serum samples were subjectedto biochemical analysis (LDH activity, GOT activity, GPT activity, CKactivity, ALP activity, AMY activity, LAP activity, LIP activity, T-CHOconcentration, F-CHO concentration, LDL-CHO concentration, HDL-CHOconcentration, TG concentration, PL concentration, GLU concentration, GA%, UA concentration, BUN concentration, CREA concentration, T-BILconcentration, D-BIL concentration, TP concentration, ALB concentration,A/G ratio, IP concentration, Ca concentration, Mg concentration, Naconcentration, K concentration, Cl concentration, Fe concentration, UIBCconcentration, and TIBC concentration). As a result, the values obtainedwith the use of the USmFZD1crd-hFcm KI chimeric mice were notsignificantly different from those of the control mice.

16-2. Tibial Bone Morphometry Using 15-Week-Old USmFZD1crd-hFcm KIChimeric Mouse 16-2-1. Bone Morphometry

In order to obtain the data regarding the mineral apposition rate, themineralization surface, and the bone formation rate, calcein (ProductNumber: 340-00433, Dojindo Laboratories, Japan) was dissolved in anaqueous solution of 2% sodium bicarbonate (Product Number: 37116-00,Kanto Chemical Co., Inc., Japan), and the prepared calcein solution (acalcium chelator) was administered subcutaneously at a dose of 16 mg/kgprior to necropsy. Administration was carried out 6 days and 1 daybefore necropsy. Tibiae were sampled at necropsy, samples ofundemineralized tibial sections were prepared, and the samples were thensubjected to toluidine blue staining (TB staining), alkaline phosphatasestaining (ALP staining), and tartrate-resistant acid phosphatasestaining (TRAP staining). In order to prepare section samples, the tibiasamples were embedded in GMA (glycolmethacrylate) resin in advance. Themetaphyseal secondary cancellous bones of the obtained samples ofundemineralized sections were subjected to measurement of the bonevolume/tissue volume as the bone structure parameter (BV/TV), theosteoblast number/bone perimeter as the bone formation parameter(Ob.N/B.Pm), the osteoblast surface/bone surface (Ob.S/BS), theosteoid/bone volume (OV/BV), the mineral apposition rate (MAR), themineralization surface/bone surface (MS/BS), the bone formationrate/bone surface (BFR/BS), the osteoclast number/bone perimeter as thebone absorption parameter (Oc.N/B.Pm), and the osteoclast surface/bonesurface (Oc.S/BS). In Example 16, all the control data were obtainedfrom 16-week-old mice.

16-2-2. Bone Volume/Tissue Volume

As a result of the measurement of the bone volume/tissue volume (BV/TV)of tibia samples obtained from 6 female control mice and 6 femaleUSmFZD1crd-hFcm KI chimeric mice subjected to necropsy, increases wereobserved in the bone volume/tissue volume of the group ofUSmFZD1crd-hFcm KI chimeric mice compared with the control group. Thissuggests the possibility that the increased bone volume/tissue volume inthe secondary cancellous bone of the tibial metaphyseal end was inducedby overexpression of the fusion protein of mouse FZD1 extracellularcysteine-rich domain and human Fc mutant.

Further, tibia samples obtained from 5 male control mice and 6 maleUSmFZD1crd-hFcm KI chimeric mice subjected to necropsy were subjected tomeasurement of the bone volume/tissue volume. As a result, the bonevolume/tissue volume of the group of USmFZD1crd-hFcm KI chimeric micewas found to have increased compared with that of the control group. Theresults demonstrate the possibility that increased bone volume/tissuevolume in the secondary cancellous bone of the tibial metaphyseal endwas induced by overexpression of the fusion protein of mouse FZD1extracellular cysteine-rich domain and human Fc mutant in male mice aswell as in female mice (Table 16).

TABLE 16 Bone volume (BV/TV) Age/sex/transgene Average 15 W ♀USmFZD1crd-hFcm KI/16 W control 15.2/4.5 15 W ♂ USmFZD1crd-hFcm KI/16 Wcontrol 11.4/5.3

16-2-3. Osteoblast Number/Bone Perimeter, Osteoblast Surface/BoneSurface, and Osteoid Volume/Bone Volume

The tibia samples obtained from 6 female control mice and 6 femaleUSmFZD1crd-hFcm KI chimeric mice subjected to necropsy were subjected tomeasurement of the osteoblast number/bone perimeter, the osteoblastsurface/bone surface, and the osteoid volume/bone volume. As a result,substantially no differences were observed between the chimeric mice andthe control mice.

Further, tibia samples obtained from 5 male control mice and 6 maleUSmFZD1crd-hFcm KI chimeric mice subjected to necropsy were subjected tomeasurement of the osteoblast number/bone perimeter, the osteoblastsurface/bone surface, and the osteoid volume/bone volume. As a result,substantially no differences were observed between the chimeric mice andthe control mice. This indicates that male mice would not be influencedby overexpression of the fusion protein of mouse FZD1 extracellularcysteine-rich domain and human Fc mutant as with the case of female mice(Table 17).

TABLE 17 Osteoblast Osteoblast Osteoid number/bone surface/bonevolume/bone perimeter surface volume (Ob. N/B. Pm) (Ob. S/BS) (OV/BV)Age/sex/transgene Average Average Average 15 W♀ USmFZD1crd- 1167/119714.4/15.2 1.08/1.28 hFcm KI/16 W control 15 W♂ USmFZD1crd- 612.6/626    8/7.5  0.1/0.38 hFcm KI/16 W control

16-2-4. Mineral Apposition Rate, Mineralization Surface, and BoneFormation Rate

As a result of measurement of the mineral apposition rate, themineralization surface, and the bone formation rate of the tibia samplesobtained from 6 female control mice and 6 female USmFZD1crd-hFcm KIchimeric mice subjected to necropsy, increases were observed in themineral apposition rate, the mineralization surface, and the boneformation rate of the group of USmFZD1crd-hFcm KI chimeric mice comparedwith the control group. This indicates that mineralization of thesecondary cancellous bone of the tibial metaphyseal end may have beenaccelerated by overexpression of the fusion protein of mouse FZD1extracellular cysteine-rich domain and human Fc mutant.

As a result of measurement of the mineral apposition rate, themineralization surface, and the bone formation rate of the tibia samplesobtained from 5 male control mice and 6 male USmFZD1crd-hFcm KI chimericmice subjected to necropsy, further, increases were observed in thechimeric mice in all items compared with control mice. This indicatesthat mineralization of the secondary cancellous bone of the tibialmetaphyseal end may have been accelerated by overexpression of thefusion protein of mouse FZD1 extracellular cysteine-rich domain andhuman Fc mutant as with the case of femal mice (Table 18).

TABLE 18 Mineral Bone formation apposition Mineralization rate/bone ratesurface/bone surface (MAR) surface (MS/BS) (BFR/BS) Age/sex/transgeneAverage Average Average 15 W♀ USmFZD1crd- 1.4/1.2 21.2/17.1 10.7/7.9 hFcm KI/16 W control 15 W♂ USmFZD1crd- 1.1/0.9 17.5/11.3 7.5/3.8 hFcmKI/16 W control

16-2-5. Osteoclast Number/Bone Perimeter and Osteoclast Surface/BoneSurface

As a result of measurement of the osteoclast number/bone perimeter andthe osteoclast surface/bone surface of the tibia samples obtained from 6female control mice and 6 female USmFZD1crd-hFcm KI chimeric micesubjected to necropsy, the values of the chimeric mice weresubstantially equivalent to those of the control mice. This indicatesthat the osteoclast number/bone perimeter and the osteoclastsurface/bone surface of the secondary cancellous bone of the tibialmetaphyseal end may not be substantially influenced by overexpression ofthe fusion protein of mouse FZD1 extracellular cysteine-rich domain andhuman Fc mutant.

As a result of measurement of the osteoclast number/bone perimeter andthe osteoclast surface/bone surface of the tibia samples obtained from 5male control mice and 6 male USmFZD1crd-hFcm KI chimeric mice subjectedto necropsy, further, the values of the chimeric mice were substantiallyequivalent to those of the control mice. This indicates that theosteoclast number/bone perimeter and the osteoclast surface/bone surfaceof the secondary cancellous bone of the tibial metaphyseal end may notbe influenced by overexpression of the fusion protein of mouse FZD1extracellular cysteine-rich domain and human Fc mutant as with the caseof female mice (Table 19).

TABLE 19 Osteoclast Osteoclast number/bone perimeter surface/bonesurface (Oc. N/B. Pm) (OC. S/BS) Age/sex/transgene Average Average 15 W♀USmFZD1crd- 169.9/181.8 2.7/1.9 hFcm KI/16 W control 15 W♂ USmFZD1crd-112.9/112.9 1.5/1.4 hFcm KI/16 W control

16-3. Measurement of Bone Strength

The femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom 5 male control mice and 6 male USmFZD1crd-hFcm KI chimeric micesubjected to necropsy, the measured values were found to have increasedin the group of USmFZD1crd-hFcm KI chimeric mice compared with thecontrol group. This indicates that the increased maximum load of thefemur may have been induced by overexpression of the fusion protein ofmouse FZD1 extracellular cysteine-rich domain-human Fc mutant (Table20).

TABLE 20 Maximum load (N) Age/sex/transgene Average 15 W♂USmFZD1crd-hFcm KI/16 W control 33/22.8

16-4. Analysis of Bone Structure of 15-Week-Old USmFZD1crd-hFcm KIChimeric Mice (3-Dimensional Microfocus X-Ray CT)

The femur samples were obtained at necropsy, and the internal structureof the cancellous bone region of the distal femoral metaphysis wasobserved using a high-resolution microfocus X-ray CT scanner (micro-CT,Scan Xmate-L090, Comscantecno Co., Ltd., Japan) and the analyticsoftware (TRY 3D-BON, Ratoc System Engineering Co., Ltd., Japan) in anon-invasive manner. The bone volume/tissue volume (BV/TV), thetrabecular thickness (Tb. Th), the trabecular number (Tb. N), thetrabecular separation (Tb. Sp), and trabecular spacing (Tb. Spac) weremeasured.

The internal structure of the cancellous bone of the femur samplesobtained from control mice (6 female mice and 6 male mice) andUSmFZD1crd-hFcm KI chimeric mice (6 female mice and 6 male mice) wasobserved via micro CT. As a result, the average bone volume/tissuevolume, trabecular thickness, and trabecular number were found to haveincreased, and the average trabecular separation and trabecular spacingwere found to have decreased in the group of USmFZD1crd-hFcm KI chimericmice compared with the control group. This suggests that the increasedbone volume/tissue volume, the increased trabecular thickness, theincreased trabecular number, the decreased trabecular separation, andthe decreased trabecular spacing in the cancellous bone of the distalfemoral metaphysis may have been caused by overexpression of the fusionprotein of mouse FZD1 extracellular cysteine-rich domain and human Fcmutant (Table 21).

TABLE 21 Bone Trabecular Trabecular volume/tissue thickness TrabecularTrabecular spacing volume (Tb. Th, number separation (Tb. Spac, (BV/TV,%) μm) (Tb. N, 1/mm) (Tb. Sp, μm) μm) Age/sex/transgene Average AverageAverage Average Average 15 W♀ 14.5/5 41.3/32.5 3.4/1.5 269.2/797.4310.5/830 USmFZD1crd-hFcm KI/16 W control 15 W♂ 13.9/6 37.4/29.4 3.5/2  260.7/510.6 298.1/540 USmFZD1crd-hFcm KI/16 W control

Example 17 17-1. Confirmation of Expression of Fusion Construct of HumanFZD1 Extracellular Cysteine-Rich Domain and Human Fc Mutant in 8- and12-Week-Old UShFZD1crd-hFcm KI Chimeric Mice

The fusion of human FZD1 extracellular cysteine-rich domain and human Fcmutant in the serum samples of the 8-week-old UShFZD1crd-hFcm KIchimeric mice (6 female mice and 6 male mice), the 8-week-old controlmice (6 female mice and 6 male mice), the 12-week-old UShFZD1crd-hFcm KIchimeric mice (6 male mice), and the 12-week-old control mice (6 malemice) prepared in accordance with the method described in Example 6 wasdetected via ELISA in accordance with the method described in Example 2.Mice were raised while humidity, temperature, and light conditions werekept constant (temperature: 22° C.; humidity: 55%; and 12 hours lightand 12 hours darkness) where they were allowed to freely eat feeds(CE-2, CLEA Japan, Inc.).

As a result, the average concentration among the 8-week-old female micewas found to be 525.5 μg/ml, that among the 8-week-old male mice wasfound to be 492.8 μg/ml, that among the 12-week-old male mice was foundto be 452.8 μg/ml, and the concentrations assayed with the use of theserum samples obtained from all control mice were lower than thedetection limit.

The above results suggest that the fusion protein of human FZD1extracellular cysteine-rich domain and human Fc mutant is expressed inmouse bodies and circulated in the blood at age of 8 weeks.

17-2. Necropsy Finding of 8-Week-Old UShFZD1crd-hFcm KI Chimeric Mouse

The chimeric mice prepared in Example 6 were subjected to necropsy atage of 8 weeks (6 female mice and 6 male mice), and the spleen, thefemur, the sternum, the cranium, the spondylus, and the costa wereobserved. As a result, whitening of the femur, whitening of the sternum,whitening and hardening of the cranium, and hardening of the costa wereobserved as characteristic changes in the UShFZD1crd-hFcm KI chimericmice compared with the control mice. In addition, a certain degree ofspleen enlargement was observed in the UShFZD1crd-hFcm KI chimeric mice.The number of mice exhibiting changes is described below.

17-2-1. Necropsy Finding of Femur

Whitening was observed in 5 mice and a certain degree of whitening wasobserved in 6 mice among the 12 UShFZD1crd-hFcm KI chimeric micesubjected to necropsy compared with the control group (6 female mice and6 male mice).

17-2-2. Necropsy Finding of Sternum

Whitening was observed in 11 mice among the 12 UShFZD1crd-hFcm KIchimeric mice subjected to necropsy compared with the control group (6female mice and 6 male mice).

17-2-3. Necropsy Finding of Cranium

Whitening was observed in 9 mice, a certain degree of whitening wasobserved in 3 mice, hardening was observed in 2 mice, and a certaindegree of hardening was observed in 5 mice among the 12 UShFZD1crd-hFcmKI chimeric mice subjected to necropsy compared with the control group(6 female mice and 6 male mice).

17-2-4. Necropsy Finding of Costa

A certain degree of hardening was observed in 5 mice among the 12UShFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with thecontrol group (6 female mice and 6 male mice).

17-2-5. Necropsy Finding of Spleen

A tendency toward enlargement was observed in 7 mice and a certaindegree of blackening was observed in 8 mice among the 12 UShFZD1crd-hFcmKI chimeric mice subjected to necropsy compared with the control group(6 female mice and 6 male mice).

The above results indicate that whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, whitening and hardeningof the spondylus, and hardening of the costa may have been induced byoverexpression of the fusion protein of humanZD1 extracellularcysteine-rich domain and human Fc mutant.

17-3. Measurement of Bone Strength of 8-Week-Old UShFZD1crd-hFcm KIChimeric Mouse

The femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom 12 control mice and 12 UShFZD1crd-hFcm KI chimeric mice, themeasured values were found to have increased in both female and malemice in the group of UShFZD1crd-hFcm KI chimeric mice compared with thecontrol group. This indicates that the increased maximum load of thefemur may have been caused by overexpression of the fusion protein ofhuman FZD1 extracellular cysteine-rich domain and human Fc mutant (Table22).

TABLE 22 Maximum load (N) Age/sex/transgene Average 8 W♀ UShFZD1crd-hFcmKI/Controls 23.8/19.3 8 W♂ UShFZD1crd-hFcm KI/Controls 27.6/19.8

17-4. Analysis of Bone Structure of 8-Week-Old UShFZD1crd-hFcm KIChimeric Mouse (3-Dimensional Microfocus X-Ray CT)

The femur samples were obtained at necropsy, and the internal structureof the cancellous bone region of the distal femoral metaphysis wasobserved using a high-resolution microfocus X-ray CT scanner (micro-CT,Scan Xmate-L090, Comscantecno Co., Ltd., Japan) and the analyticsoftware (TRY 3D-BON, Ratoc System Engineering Co., Ltd., Japan) in anon-invasive manner. The bone volume/tissue volume (BV/TV), thetrabecular thickness (Tb. Th), the trabecular number (Tb. N), thetrabecular separation (Tb. Sp), and the trabecular spacing (Tb. Spac)were measured.

The internal structures of the cancellous bones of the femur samplesobtained from control mice (6 female mice and 6 male mice) andUShFZD1crd-hFcm KI chimeric mice (6 female mice and 6 male mice) wereobserved via micro CT. As a result, the average bone volume/tissuevolume, trabecular thickness, and trabecular number were found to haveincreased, and the average trabecular separation and trabecular spacingwere found to have decreased in the group of UShFZD1crd-hFcm KI chimericmice compared with the control group. This suggests that the increasedvolume/tissue volume, the increased trabecular thickness, the increasedtrabecular number, the decreased trabecular separation, and thedecreased trabecular spacing in the cancellous bone of the distalfemoral metaphysis may have been caused by overexpression of the fusionprotein of human FZD1 extracellular cysteine-rich domain and human Fcmutant (Table 23).

TABLE 23 Bone Trabecular Trabecular Trabecular volume/tissue thicknessnumber Trabecular spacing volume (Tb. Th, (Tb. N, separation (Tb. Spac,(BV/TV, %) μm) 1/mm) (Tb. Sp, μm) μm) Age/sex/transgene Average AverageAverage Average Average 8 W♀ 20.8/7.6   41/28.2 5.05/2.6  159.1/367.5200.2/395.7 UShFZD1crd-hFcm KI/8 W control 8 W♂ 17.3/8.6 35.2/28.4 4.8/3.02 170.4/312.1 205.6/340.6 UShFZD1crd-hFcm KI/8 W control

17-5. Necropsy Finding of 12-Week-Old UShFZD1crd-hFcm KI Chimeric Mouse

The chimeric mice prepared in Example 6 (6 male mice) were subjected tonecropsy at age of 12 weeks, and the spleen, the femur, the sternum, thecranium, the spondylus, and the costa were observed. As a result,whitening of the femur, whitening of the sternum, whitening andhardening of the cranium, and a certain degree of hardening of the costawere observed as characteristic changes in the UShFZD1crd-hFcm KIchimeric mice compared with the control mice (6 male mice). The numberof mice exhibiting changes is described below.

17-5-1. Necropsy Finding of Femur

A certain degree of whitening was observed in 5 mice among the 6UShFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with thecontrol group (6 mice).

17-5-2. Necropsy Finding of Sternum

Whitening was observed in 3 mice and a certain degree of whitening wasobserved in 3 mice among the 6 UShFZD1crd-hFcm KI chimeric micesubjected to necropsy compared with the control group (6 mice).

17-5-3. Necropsy Finding of Cranium

Whitening was observed in a mouse, a certain degree of whitening wasobserved in 4 mice, hardening was observed in a mouse, and a certaindegree of hardening was observed in 2 mice among the 6 UShFZD1crd-hFcmKI chimeric mice subjected to necropsy compared with the control group(6 mice).

17-5-4. Necropsy Finding of Costa

A certain degree of hardening was observed in 2 mice among the 6UShFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with thecontrol group (6 mice).

The above results indicate that whitening of the femur, whitening of thesternum, whitening and hardening of the cranium, and a certain degree ofhardening of the costa may have been caused by overexpression of thefusion protein of human FZD1 extracellular cysteine-rich domain andhuman Fc mutant.

17-6. Pathological Finding of 12-Week-Old UShFZD1crd-hFcm KI ChimericMouse

The H&E stained femur and sternum pathological sections obtained fromsix 12-week-old control chimeric mice and six UShFZD1crd-hFcm KIchimeric mice were observed. As a result, the thickened femoraldiaphyseal wall (FIGS. 19 and 20, Table 24), the increased cancellousbone (FIG. 21), and the increased sternal cancellous bone (FIG. 22) wereobserved in the UShFZD1crd-hFcm KI chimeric mice compared with controlmice. The number of mice exhibiting changes is described below.

17-6-1. Femur

The increased cancellous bone was observed in all the 6 UShFZD1crd-hFcmKI chimeric mice subjected to necropsy compared with 6 control mice.Further, transected sections obtained from 3 femoral sites (i.e., sites30%, 50%, and 80% away from the proximal end) were subjected tomeasurement of the diaphyseal wall thickness. As a result, the averageminimum wall thickness at a site 30% away from the proximal end and theaverage maximum wall thickness at a site 50% away from the proximal endwere found to be larger than those of the control group (FIGS. 19 and20, Table 24).

TABLE 24 Minimum diaphyseal Maximum diaphyseal wall thickness at sitewall thickness at site 30% away from 50% away from proximal end (mm)proximal end (mm) Age/sex/transgene Average Average 12 W♂ UShFZD1crd-0.21/0.19 0.47/0.34 hFcm KI/12 W control

17-6-2. Sternum

The increased cancellous bone was observed in all of the 6UShFZD1crd-hFcm KI chimeric mice subjected to necropsy compared with 6control mice (FIG. 22).

The above results demonstrate that the thickened femoral diaphysealwall, the increased cancellous bone, and the increased sternalcancellous bone may have been caused by overexpression of the fusionprotein of human FZD1 extracellular cysteine-rich domain and human Fcmutant.

17-7. Biochemical Analysis of Serum

Six 12-week-old UShFZD1crd-hFcm KI male chimeric mice and 6 male controlmice were exsanguinated under ether anesthesia to prepare serum samples.With the use of Hitachi 7180 (Hitachi Science Systems Ltd., Japan),serum samples were subjected to biochemical analysis (LDH activity, GOTactivity, GPT activity, CK activity, ALP activity, AMY activity, LAPactivity, LIP activity, T-CHO concentration, F-CHO concentration,LDL-CHO concentration, HDL-CHO concentration, TG concentration, PLconcentration, GLU concentration, GA %, UA concentration, BUNconcentration, CREA concentration, T-BIL concentration, D-BILconcentration, TP concentration, ALB concentration, A/G ratio, IPconcentration, Ca concentration, Mg concentration, Na concentration, Kconcentration, Cl concentration, Fe concentration, UIBC concentration,and TIBC concentration). As a result, the values obtained with the useof the UShFZD1crd-hFcm KI chimeric mice were not significantly differentfrom those of the control mice.

Example 18

The calvarial thickness of the cranium and the bone strength of thefemur obtained from the mice to which the mFZD1crd-hFcm recombinant hadbeen administered (described in Example 12) were measured.

18-1. Measurement of Calvarial Thickness of Cranium

Toluidine blue-stained cranium samples were prepared, and the calvarialthickness (μm) of the outer surface of the cranium to the parietaltemporal suture (the squamous border) was measured while excluding theareas 0.6 mm each to the right and the left of the sagittal suture.

As a result of measurement of the calvarial thickness using 5 femalecontrol mice and 5 female mice to which the mFZD1crd-hFcm recombinanthad been administered, the value of the mice to which the mFZD1crd-hFcmrecombinant had been administered (average: 219.4 μm) was found to haveincreased compared with that of the control group (average: 213.9 μm).This suggests that the increased calvarial thickness of the cranium mayhave been caused by administration of the recombinant mFZD1crd-hFcm.

18-2. Measurement of Bone Strength of Femur

The femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom 5 female control mice and 5 female mice to which the recombinantmFZD1crd-hFcm had been administered, the measured values of the group ofmice to which the recombinant mFZD1crd-hFcm had been administered werefound to have increased (average: 31.3 N) compared with the values ofthe control group (average: 26.2 N). This indicates that the increasedmaximum load of the femur may have been caused by administration of therecombinant mFZD1crd-hFcm.

Example 19

Analysis Using USmFZD2crd-hFcm KI Chimeric Mouse Prepared in Accordancewith the Method Described in Example 9

19-1. Blood Cell Analysis

Six 8-week-old USmFZD2crd-hFcm KI female chimeric mice, six 8-week-oldUSmFZD2crd-hFcm KI male chimeric mice, eleven 8-week-old female controlmice, eleven 8-week-old male control mice, six 15-week-oldUSmFZD2crd-hFcm KI female chimeric mice, six 15-week-old USmFZD2crd-hFcmKI male chimeric mice, eleven 15-week-old female control mice, andeleven 15-week-old male control mice were subjected to orbital bloodsampling using a glass capillary under ether anesthesia, and theobtained blood samples were subjected to blood component analysis usingADVIA120 (Bayer Medical Ltd., Japan) (blood components: erythrocytecounts, hemoglobin, hematocrit, MCH, MCHC, reticulocyte counts,leukocyte counts, blood platelet counts, lymphocyte counts, neutrophilcounts, monocyte counts, eosinophil counts, and basophil counts). As aresult, the values obtained with the use of the USmFZD2crd-hFcm KIchimeric mice were not significantly different from those of the controlmice at ages of 8 weeks and 15 weeks.

19-2. Biochemical Analysis of Serum

Six USmFZD2crd-hFcm KI female chimeric mice, 6 USmFZD7crd-hFcm KI malechimeric mice, 9 female control mice, and 9 male control mice at age of16 weeks were exsanguinated under ether anesthesia to prepare serumsamples. With the use of Hitachi 7180 (Hitachi Science Systems Ltd.,Japan), serum samples were subjected to biochemical analysis (LDHactivity, GOT activity, GPT activity, CK activity, ALP activity, AMYactivity, LAP activity, LIP activity, T-CHO concentration, F-CHOconcentration, LDL-CHO concentration, HDL-CHO concentration, TGconcentration, PL concentration, GLU concentration, GA %, UAconcentration, BUN concentration, CREA concentration, T-BILconcentration, D-BIL concentration, TP concentration, ALB concentration,A/G ratio, IP concentration, Ca concentration, Mg concentration, Naconcentration, K concentration, Cl concentration, Fe concentration, UIBCconcentration, and TIBC concentration). As a result, the values obtainedwith the use of the USmFZD2crd-hFcm KI chimeric mice were notsignificantly different from those of the control mice.

19-3. Confirmation of Expression of the Fusion Protein of Mouse FZD2Extracellular Cysteine-Rich Domain and Human Fc Mutant inUSmFZD2crd-hFcm KI Chimeric Mouse

The concentration of the fusion protein of mouse FZD2 extracellularcysteine-rich domain and human Fc mutant existing in the serum samplesof the 16-week-old USmFZD2crd-hFcm KI chimeric mice (6 female mice and 6male mice) prepared in accordance with the method described in Example 2was detected via ELISA. Mice were raised while humidity, temperature,and light conditions were kept constant (temperature: 22° C.; humidity:55%; and 12 hours light and 12 hours darkness) where they were allowedto freely eat feeds (CE-2, CLEA Japan, Inc.).

As a result, the average concentration among the 16-week-old femaleUSmFZD2crd-hFcm KI chimeric mice was found to be 31.5 μg/ml, that amongthe 16-week-old male mice was found to be 26.2 μg/ml, and theconcentrations assayed with the use of control mice (6 female mice and 6male mice) were lower than the detection limit.

The above results suggest that the fusion protein of FZD2 extracellularcysteine-rich domain and human Fc mutant is expressed in mouse bodiesand circulated in the blood.

19-4. Tibial Bone Morphometry Using 18-Week-Old USmFZD2crd-hFcm KIChimeric Mouse 19-4-1. Bone Morphometry

In order to obtain the data regarding the mineral apposition rate, themineralization surface, and the bone formation rate, calcein (ProductNumber: 340-00433, Dojindo Laboratories, Japan) was dissolved in anaqueous solution of 2% sodium bicarbonate (Product Number: 37116-00,Kanto Chemical Co., Inc., Japan), and the prepared calcein solution (acalcium chelator) was administered subcutaneously at a dose of 16 mg/kgprior to necropsy. Administration was performed 6 days and 1 day beforenecropsy. Tibiae were sampled at necropsy, the samples ofundemineralized tibial sections were prepared, and the samples were thensubjected to toluidine blue staining (TB staining), alkaline phosphatasestaining (ALP staining), and tartrate-resistant acid phosphatasestaining (TRAP staining). In order to prepare section samples, the tibiasamples were embedded in GMA (glycolmethacrylate) resin in advance. Themetaphyseal secondary cancellous bones of the obtained samples ofundemineralized sections were subjected to measurement of the bonevolume/tissue volume as the bone structure parameter (BV/TV), theosteoblast number/bone perimeter as the bone formation parameter(Ob.N/B.Pm), the osteoblast surface/bone surface (Ob.S/BS), theosteoid/bone volume (OV/BV), the mineral apposition rate (MAR), themineralization surface/bone surface (MS/BS), the bone formationrate/bone surface (BFR/BS), the osteoclast number/bone perimeter as thebone absorption parameter (Oc.N/B.Pm), and the osteoclast surface/bonesurface (Oc.S/BS). In Example 19-2 and subsequent examples, all thecontrol data were obtained from 16-week-old mice.

19-4-2. Bone Volume/Tissue Volume

As a result of the measurement of the bone volume/tissue volume of tibiasamples obtained from 6 female control mice and 3 female USmFZD2crd-hFcmKI chimeric mice subjected to necropsy, increases were observed in thebone volume/tissue volume of the group of USmFZD2crd-hFcm KI chimericmice compared with the control group. This suggests the possibility thatthe increased bone volume/tissue volume of the secondary cancellous boneof the tibial metaphyseal end was caused by overexpression of the fusionprotein of mouse FZD2 extracellular cysteine-rich domain and human Fcmutant.

Further, tibia samples obtained from 5 male control mice and 4 maleUSmFZD2crd-hFcm KI chimeric mice subjected to necropsy were subjected tomeasurement of the bone volume/tissue volume. As a result, the bonevolume/tissue volume of the group of USmFZD2crd-hFcm KI chimeric micewas found to have increased compared with that of the control group. Theresults demonstrate the possibility that increased bone volume/tissuevolume in the secondary cancellous bone of the tibial metaphyseal endwas induced by overexpression of the mouse FZD2 extracellularcysteine-rich domain-human Fc mutant fusion constructs in male mice aswell as in female mice (Table 25).

TABLE 25 Bone volume/tissue Age/sex/transgene volum (BV/TV) Average 18W♀ USmFZD2crd-hFcm KI/16 W control 15.3/4.5 18 W♂ USmFZD2crd-hFcm KI/16W control 14.9/5.3

19-4-3. Osteoblast Number/Bone Perimeter, Osteoblast Surface/BoneSurface, and Osteoid/Bone Volume

The osteoblast number/bone perimeter, the osteoblast surface/bonesurface, and the osteoid/bone volume were measured using the tibiasamples obtained from 6 female control mice and 3 female USmFZD2crd-hFcmKI chimeric mice subjected to necropsy. As a result, substantially nodifferences were observed between the chimeric mice and the controlmice.

Further, the osteoblast number/bone perimeter, the osteoblastsurface/bone surface, and the osteoid/bone volume were measured usingthe tibia samples obtained from 5 male control mice and 4 maleUSmFZD2crd-hFcm KI chimeric mice subjected to necropsy. As a result,substantially no differences were observed between the chimeric mice andthe control mice. This indicates that male mice would not be influencedby overexpression of the fusion protein of mouse FZD2 extracellularcysteine-rich domain and human Fc mutant as with the case of female mice(Table 26).

TABLE 26 Osteoblast Osteoblast Osteoid number/bone surface/bonevolume/bone perimeter surface volume (Ob. N/B. Pm) (Ob. S/BS) (OV/BV)Age/sex/transgene Average Average Average 18 W♀ USmFZD2crd- 1257.7/119715.5/15.2 1.3/1.28 hFcm KI/16 W control 18 W♂ USmFZD2crd- 566.1/6266.1/7.5 0.2/0.38 hFcm KI/16 W control

19-4-4. Mineral Apposition Rate, Mineralization Surface, and BoneFormation Rate

As a result of measurement of the mineral apposition rate, themineralization surface, and the bone formation rate of the tibia samplesobtained from 6 female control mice and 3 female USmFZD2crd-hFcm KIchimeric mice subjected to necropsy, increases were observed in themineral apposition rate, the mineralization surface, and the boneformation rate of the group of USmFZD2crd-hFcm KI chimeric mice comparedwith the control group. This indicates that mineralization of thesecondary cancellous bone of the tibial metaphyseal end may have beenaccelerated by overexpression of the fusion protein of mouse FZD2extracellular cysteine-rich domain and human Fc mutant.

As a result of measurement of the mineral apposition rate, themineralization surface, and the bone formation rate of the tibia samplesobtained from 5 male control mice and 4 male USmFZD1crd-hFcm KI chimericmice subjected to necropsy, further, increases was observed in themineralization surface compared with the control group (Table 27).

TABLE 27 Bone Mineralization formation Mineral surface/bone rate/boneapposition rate surface surface (MAR) (MS/BS) (BFR/BS) Age/sex/transgeneAverage Average Average 18 W♀ USmFZD2crd- 1.7/1.2 22.7/17.1 14.1/7.9 hFcm KI/16 W control 18 W♂ USmFZD2crd- 0.8/0.9 13.4/11.3 4.2/3.8 hFcmKI/16 W control

19-4-5. Osteoclast Number/Bone Perimeter and Osteoclast Surface/BoneSurface

As a result of measurement of the osteoclast number/bone perimeter andthe osteoclast surface/bone surface of the tibia samples obtained from 6female control mice and 3 female USmFZD2crd-hFcm KI chimeric micesubjected to necropsy, both values were found to tend to decreasecompared with the control group. This indicates that the osteoclastnumber/bone perimeter and the osteoclast surface/bone surface of thesecondary cancellous bone of the tibial metaphyseal end may have beensuppressed by overexpression of the fusion protein of mouse FZD2extracellular cysteine-rich domain and human Fc mutant.

As a result of measurement of the osteoclast number/bone perimeter andthe osteoclast surface/bone surface of the tibia samples obtained from 5male control mice and 4 male USmFZD2crd-hFcm KI chimeric mice subjectedto necropsy, both values were found to tend to decrease compared withthe control group. This indicates that the osteoclast number/boneperimeter and the osteoclast surface/bone surface of the secondarycancellous bone of the tibial metaphyseal end may have been suppressedby overexpression of the fusion protein of mouse FZD2 extracellularcysteine-rich domain and human Fc mutant as with the case of female mice(Table 28).

TABLE 28 Osteoclast Osteoclast surface/bone number/bone perimete surface(Oc. N/B. Pm) (OC. S/BS) Age/sex/transgene Average Average 18 W♀USmFZD2crd- 112.6/181.8 1.6/1.9 hFcm KI/16 W control 18 W♂ USmFZD2crd- 67.4/112.9 0.9/1.4 hFcm KI/16 W control

19-5. Measurement of Bone Strength

The femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom six 16-week-old female control mice and three 18-week-oldUSmFZD2crd-hFcm KI chimeric mice subjected to necropsy, the measuredvalues were found to have increased in the group of USmFZD2crd-hFcm KIchimeric mice compared with the control group. This indicates that theincreased maximum load of the femur may have been caused byoverexpression of the fusion protein of mouse FZD2 extracellularcysteine-rich domain and human Fc mutant.

As a result of measurement of the maximum load of femur samples obtainedfrom five 16-week-old male control mice and four 18-week-old maleUSmFZD2crd-hFcm KI chimeric mice subjected to necropsy, further, themeasured values were found to have increased in the group ofUSmFZD2crd-hFcm KI chimeric mice compared with the control group. Thisindicates that the increased maximum load of the femur may have beeninduced by overexpression of the fusion protein of mouse FZD2extracellular cysteine-rich domain and human Fc mutant as with the caseof female mice (Table 29).

TABLE 29 Maximum load (N) Age/sex/transgene Average 18 W♀USmFZD2crd-hFcm KI/16 W control 32.6/26.6 18 W♂ USmFZD2crd-hFcm KI/16 Wcontrol 30.3/22.8

19-6. Analysis of Bone Structure of 18-Week-Old USmFZD2crd-hFcm KIChimeric Mouse (3-Dimensional Microfocus X-Ray CT)

The femur samples were obtained at necropsy, and the internal structureof the cancellous bone region of the distal femoral metaphysis wasobserved using a high-resolution microfocus X-ray CT scanner (micro-CT,Scan Xmate-L090, Comscantecno Co., Ltd., Japan) and the analyticsoftware (TRY 3D-BON, Ratoc System Engineering Co., Ltd., Japan) in anon-invasive manner. The bone volume/tissue volume (BV/TV), thetrabecular thickness (Tb. Th), the trabecular number (Tb. N), thetrabecular separation (Tb. Sp), and trabecular spacing (Tb. Spac) weremeasured.

The internal structure of the cancellous bone of the femur samplesobtained from 16-week-old control mice (6 female mice and 6 male mice)and 18-week-old USmFZD2crd-hFcm KI chimeric mice (3 female mice and 4male mice) was observed via micro CT. The average bone volume/tissuevolume, trabecular thickness, and trabecular number were found to haveincreased, and the average trabecular separation and trabecular spacingwere found to have decreased in the group of USmFZD2crd-hFcm KI chimericmice compared with the control group. This suggests that the increasedbone volume/tissue volume, the increased trabecular thickness, theincreased trabecular number, the decreased trabecular separation, andthe decreased trabecular spacing in the cancellous bone of the distalfemoral metaphysis may have been caused by overexpression of the fusionprotein of mouse FZD2 extracellular cysteine-rich domain and human Fcmutant (Table 30).

TABLE 30 Bone Trabecular Trabecular Trabecular Trabecular volume/tissuethickness number separation spacing volume (Tb. Th, (Tb. N, (Tb. Sp,(Tb. Spac, (BV/TV, %) μm) 1/mm) μm) μm) Age/sex/transgene AverageAverage Average Average Average 18 W♀ USmFZD2crd-hFcm 17.2/5 43.1/32.53.9/1.5 209.7/797.4 252.8/830 KI/16 W control 18 W♂ USmFZD2crd-hFcm15.2/6 40.2/29.4 3.7/2   226.6/510.6 266.8/540 KI/16 W control

Example 20

Expression and Preparation of Recombinant mFZD2crd-hFcm20-1. Construction of Recombinant mFZD2crd-hFcm Expression Vector

The recombinant mFZD2crd-hFcm expression vector was constructed usingthe PCR primers shown in SEQ ID NOs: 54, 66, 67, and 68 and, astemplates, mouse FZD2 cDNA (SEQ ID NO: 58) and hFcm cDNA (SEQ ID NO: 3)in accordance with the method described in Example 7-1 (FIG. 23).

20-1-1. Construction of pLN1V5 Vector

Sense oligo DNA (V5S) having the BamHI, NheI, and SalI sites at the 5′terminus and the XhoI site at the 3′ terminus (a V5 tag and a stopcodon) and corresponding antisense oligo DNA (V5AS) were synthesized.

V5S: (SEQ ID NO: 50) GATCCGCTAGCGTCGACGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTGAC V5AS: (SEQ ID NO: 51)TCGAGTCACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACC GTCGACGCTAGCG

Oligo DNA synthesized above was introduced into the BamHI-XhoI site onthe pLN1 vector described in the report of Kakeda et al. (Gene Ther.,12, 852-856, 2005) to construct the pLN1V5 vector.

20-1-2. Synthesis of mFZD2crd-hFcm DNA Fragment

155Fc_BHIkozakFw:  (SEQ ID NO: 66)TAAAGGATCCCGGCCACCATGCGGGCCCGCAGCGCCCTGC  155Fc_mFZD2G1SA_3 primer: (SEQ ID NO: 67) GTCTGAAGACCTAGGCTCGGCTAGCGCAGGAGCTCCGTCC GISA_5 primer:  (SEQ ID NO: 54) GCCGAGCCTAGGTCTTCAGAC  hFc-NotI-Rv: (SEQ ID NO: 68) ATAGTTTAGCGGCCGCTCATTTACCCGGAGACAGG 

A reaction solution was prepared using Prime STAR HS DNA Polymerase(Takara Bio Inc., Japan) in accordance with the instructions, 10 pmoleach primers shown in SEQ ID NOs: 66 and 67 and mouse FZD2 cDNA (SEQ IDNO: 58) as a template were added to 50 of the reaction solution, theresultant was incubated at 98° C. for 1 minute, an amplification cycleof 98° C. for 10 seconds, 62° C. for 5 seconds, and 72° C. for 40seconds was repeated 30 times, and the resulting 543-bp amplifiedfragment was separated and recovered with 0.8% gel. The amplifiedfragment (BamHI mFZD2crd hFcm) was recovered from the gel using theQIAquick Gel Extraction Kit (Qiagen, Japan) in accordance with theinstructions.

Similarly, a reaction solution was prepared using Prime STAR HS DNAPolymerase (Takara Bio Inc., Japan) in accordance with the instructions,10 pmol each primers shown in SEQ ID NOs: 54 and 68 and hFcm cDNA (SEQID NO: 3) as a template were added to 50 μl of the reaction solution,the resultant was incubated at 98° C. for 1 minute, an amplificationcycle of 98° C. for 10 seconds, 62° C. for 5 seconds, and 72° C. for 40seconds was repeated 30 times, and the resulting 718-bp amplifiedfragment was separated and recovered with 0.8% gel. The amplifiedfragment (hFcm NotI) was recovered from the gel using the QIAquick GelExtraction Kit (Qiagen, Japan) in accordance with the instructions.

The amplified DNA fragments obtained via the two above PCR procedures(i.e., BamHI mFZD2 hFcm and hFcm NotI) were added in amounts of 10 μleach, the solution was heated at 100° C. for 3 minutes, and thetemperature was lowered to room temperature, followed by annealing ofthe hFcm region. Thereafter, 10 pmol each primers shown in SEQ ID NOs:66 and 68 were added, an extension reaction was carried out at 72° C.for 5 minutes, the resultant was heated at 98° C. for 1 minute, anamplification cycle of 98° C. for 10 seconds, 62° C. for 5 seconds, and72° C. for 1 minutes was repeated 30 times, and the resulting 1,240-bpamplified fragment was separated and recovered with 0.8% gel. Theamplified fragment was recovered from the gel using the QIAquick GelExtraction Kit (Qiagen, Japan) in accordance with the instructions.

20-1-3. Construction of mFZD2crd-hFcm Recombinant Expression Vector

The PCR-amplified fragment recovered in Example 20-1-2 was digested withthe BamHI and NotI restriction enzymes (Roche Diagnostics, K. K.,Japan), and the resultant was separated and recovered with 0.8% agarosegel. The enzyme-treated fragment was recovered from the gel using theQIAquick Gel Extraction Extraction Kit (Qiagen, Japan) in accordancewith the instructions. The NotI site was added to the pLN1V5 vectorprepared in Example 20-1-1 to prepare another vector, and the obtainedenzyme-treated fragment was introduced into the BamHI.NotI site of theresulting vector to construct the mFZD2crd-hFcm recombinant expressionvector (FIG. 23).

A polynucleotide sequence (1206 bp, SEQ ID NO: 69) comprising a regionfrom the initiation codon to the termination codon of cDNA of therecombinant mFZD2crd-hFcm and an amino acid sequence (401 amino acids,SEQ ID NO: 70) comprising the signal sequence of mFZD2-hFcm encoded bythe cDNA are shown below. In SEQ ID NOs: 69 and 70, the underlinedportion represents the mouse FZD2 signal sequence.

SEQ ID NO: 69:  ATGCGGGCCCGCAGCGCCCTGCCCCGCAGCGCCCTGCCCCGCCTGCTGCTGCCACTGCTGCTGCTGCCGGCCGCCGGACCGGCCCAGTTCCACGGGGAGAAGGGCATCTCCATCCCGGACCACGGCTTCTGCCAGCCCATCTCCATCCCGCTGTGCACGGACATCGCCTACAACCAGACCATCATGCCCAACCTTCTTGGCCACACGAACCAGGAAGACGCGGGCCTGGAGGTGCATCAGTTCTACCCGCTGGTGAAGGTGCAGTGCTCGCCCGAGCTGCGCTTCTTCCTGTGCTCCATGTACGCGCCGGTGTGCACAGTGCTGGAGCAGGCCATCCCGCCGTGCCGCTCCATCTGCGAGCGCGCGCGCCAAGGCTGCGAGGCGCTCATGAACAAGTTCGGCTTCCAATGGCCCGAGCGCCTCCGCTGCGAGCATTTCCCGCGTCACGGCGCGGAGCAGATCTGCGTGGGCCAGAACCACTCGGAGGACGGAGCTCCTGCGCTAGCCGAGCCTAGGTCTTCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATGASEQ ID NO: 70:  MRARSALPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALAEPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGICEYKCAVSNKALPASIEKTISICAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK20-2. Transient Expression of Recombinant mFZD2crd-hFcm UsingRecombinant mFZD2crd-hFcm Expression Vector

20-2-1. Preparation of Expression Vector Used for Gene Introduction

The recombinant mFZD2crd-hFcm expression vector obtained in Example20-1-3 was introduced into E. coli DH5a, and DNA was prepared from thetransformant cells using a plasmid purification kit (Qiagen plasmid Maxikit, Qiagen, Japan).

20-2-2. Introduction of Vector into Cultured Cell and SecretoryExpression

FreeStyle CHO-S cells (Invitrogen, Japan) were cultured in FreeStyle CHOexpression medium (Invitrogen, Japan) at 37° C. in the presence of 5%CO₂ at 125 rpm to reach a cell density of 1×10⁵ to 4×10⁶ cells/ml. Whenculture was conducted using 1 liter of medium, 20 ml of Opti PRO SFM(Invitrogen, Japan) was added to 1 mg of the expression vector, and 12.5ml of Opti PRO SFM (Invitrogen, Japan) was added to 7.5 ml ofpolyethyleneimine (PEI). These solutions were mixed with each otherimmediately thereafter, and the resultant was incubated at roomtemperature for 10 minutes. Thereafter, the expression vector treated inthe manner described above was added to a medium containing 2×10⁹cells/1 of FreeStyle CHO-S cells, and culture was conducted for 3 days.

20-3. Purification and Preparation of Recombinant mFZD2crd-hFcm

20-3-1. Pretreatment of Culture Supernatant

After culture, the supernatant was recovered, filtered through a 0.22 μmfilter (TC Filter Unit, PES, Nalgene), and then cooled to 4° C. (in alow-temperature chamber). When cryopreserved, the resultant was thawedand then filtered through a 0.22 μm filter again.

20-3-2. Antibody Affinity Chromatography

The acidic buffer used is 1 liter of a solution comprising 3.43 g ofcitrate monohydrate (Nakalai Tesque, Inc., Japan, MW: 210.14), 0.90 g oftrisodium citrate (Wako Pure Chemical Industries, Ltd., MW: 258.07), and8.77 g of sodium chloride (Junsei Chemical Co., Ltd., MW: 58.44)dissolved in Milli-Q water. The neutralizing buffer used is 1 liter of asolution comprising 13.1 g of sodium dihydrogen phosphate dihydrate(Kanto Chemical Co., Inc., MW: 156.01), 41.5 g of disodium hydrogenphosphate dodecahydrate (Wako Pure Chemical Industries, Ltd., MW:358.14), and 8.77 g of sodium chloride (Junsei Chemical Co., Ltd., MW:58.44) dissolved in Milli-Q water.

The pretreated culture supernatant (1 liter) was applied to a protein Acolumn (Hi Trap Protein A HP, 5 ml, GE Healthcare Bio-Sciences Corp.,Japan) equilibrated with PBS (Dulecco's phosphate buffered saline,SIGMA). Thereafter, the column was washed with 25 ml or more PBS, andthe column was washed again with 30 ml of PBS. After the completion ofthe washing procedure, 25 ml of acidic buffer was added to the column,and the target protein was recovered. AKTAexplorer 10s (GE HealthcareBio-Sciences Corp, Japan) was used in the separation and purificationprocedure. Endotoxin was removed before use.

20-3-3. Preparation of Purified Authentic Sample

The purified authentic sample obtained in Example 20-3-2 wasconcentrated using an ultrafilter membrane VIVASPIN20 10,000 MWCO PES(Sartorius Stedim Japan K. K., Japan). Thereafter, the buffer in thesample was substituted with PBS using NAP-25 Columns (GE HealthcareBio-Sciences Corp, Japan). After the completion of the concentration andsubstitution procedure, the resultanting solution was filtered through a0.22 Inn filter (Millex G V, Millipore, Japan). The concentrationprocedure was carried out in a clean bench to the extent possible. Allthe procedures conducted in Example 20-3 other than those conducted in aclean bench were carried out in a low-temperature chamber (+4° C.) or onice. A protein concentration was determined by measuring a specificabsorbance at 280 nm (A280 nm) (E1%, 1 cm=9.7).

Example 21

Expression and Preparation of Recombinant mFZD7c10-hFcm21-1. Construction of Recombinant mFZD7c10-hFcm Expression Vector

The recombinant mFZD7c10-hFcm expression vector was constructed usingthe PCR primers shown in SEQ ID NOs: 55 and 71 and, as templates, mouseFZD7 cDNA (SEQ ID NO: 1) and hFcm cDNA (SEQ ID NO: 3) in accordance withthe method described in Example 7-1 (FIG. 24).

21-1-1. Construction of pLN1V5 Vector

Sense oligo DNA (V5S) having the BamHI, NheI, and SalI sites at the 5′terminus and the XhoI site at the 3′ terminus (a V5 tag and a stopcodon) and corresponding antisense oligo DNA (V5AS) were synthesized.

V5S: (SEQ ID NO: 50) GATCCGCTAGCGTCGACGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTGAC V5AS: (SEQ ID NO: 51)TCGAGTCACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACC GTCGACGCTAGCG

Oligo DNA synthesized above was introduced into the BamHI-XhoI site onthe pLN1 vector described in the article of Kakeda et al. (Gene Ther.,12, 852-856, 2005) to construct the pLN1V5 vector.

21-1-2. Synthesis of mFZD7c10-hFcm DNA Fragment

pLN1V5-BHIkozakFw:   (SEQ ID NO: 71)TAAAGGATCCCGGCCACCATGGAGACAGACACACTCCTG   SalIG1SARv:  (SEQ ID NO: 55) TAAAGTCGACTCATTTACCCGGAGACAGGG 

A reaction solution was prepared using Prime STAR HS DNA Polymerase(Takara Bio Inc., Japan) in accordance with the instructions, 10 pmoleach primers shown in SEQ ID NOs: 71 and 55 and, as a template, thefusion DNA construct encoding the fusion protein of mouse Igκ signalsequence, Frizzled 7 mouse CRD, and mutant human IgG1-derived Fc proteinprepared in accordance with the method described in Example 1 were addedto 50 μl of the reaction solution, the mixture was incubated at 98° C.for 10 seconds, an amplification cycle of 98° C. for 10 seconds, 57° C.for 5 seconds, and 72° C. for 2 minutes was repeated 20 times, and theresulting 1,367-bp amplified fragment was separated and recovered with0.8% gel. The amplified fragment (BamHI mFZD7c10hFcm SalI) was recoveredfrom the gel using the QIAquick Gel Extraction Kit (Qiagen, Japan) inaccordance with the instructions.

21-1-3. Construction of Recombinant mFZD7c10-hFcm Expression Vector

The PCR-amplified fragment recovered in Example 21-1-2 was digested withthe BamHI and SalI restriction enzymes (Roche Diagnostics, Japan), andthe resultant was separated and recovered with 0.8% agarose gel. Theenzyme-treated fragment was recovered from the gel using the QIAquickGel Extraction Kit (Qiagen, Japan) in accordance with the instructions.The obtained enzyme-treated fragment was introduced into the BamHI.SalIsite of the pLN1V5 vector prepared in Example 21-1-1 to construct themFZD7c10-hFcm recombinant expression vector (FIG. 24).

The polynucleotide sequence (1339 bp, SEQ ID NO: 72) comprising a regionfrom the initiation codon to the termination codon of cDNA of therecombinant mFZD7c10-hFcm, and the amino acid sequence (365 amino acids,SEQ ID NO: 73) comprising the mouse Igκ signal sequence encoded by thecDNA, are shown below. In SEQ ID NOs: 72 and 73, the underlined portionrepresents the mouse Igκ signal sequence, the region marked by the solidbox represents the cysteine-rich domain comprising N-terminal cysteine 1to cysteine 10 of the mouse Frizzled 7 extracellular region protein (theminimum CRD region), and the region marked by the double underlinerepresents hFcm.

SEQ ID NO: 72:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTT

GGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO: 73:

THTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21-2. Transient Expression of Recombinant mFZD7c10-hFcm UsingRecombinant mFZD7c10-hFcm Expression Vector

21-2-1. Preparation of Expression Vector Used for Gene Introduction

The recombinant mFZD7c10-hFcm expression vector obtained in Example21-1-3 was introduced into E. coli DH5a, and DNA was prepared from thetransformant using a plasmid purification kit (Qiagen plasmid Maxi kit,Qiagen, Japan).

21-2-2. Introduction of Vector into Cultured Cell and SecretoryExpression

FreeStyle 293F cells (Invitrogen Japan K. K.) are cultured in FreeStyle293 expression medium (Invitrogen Japan K. K.) at 37° C. in the presenceof 5% CO₂ at 125 rpm to reach a cell density of 2×10⁵ to 3×10⁶ cells/ml.When culture was conducted using 1 liter of medium, 35 ml of theOpti-MEM I reduced serum medium (Invitrogen, Japan) was added to 1 mg ofthe expression vector, 33.7 ml of the Opti-MEM I reduced serum medium(Invitrogen, Japan) was added to 1.3 ml of the 293 fectin transfectionreagent (Invitrogen, Japan), and the resulting solutions were incubatedat room temperature for 5 minutes. These solutions were mixed with eachother after incubation, and the resultant was further incubated at roomtemperature for 20 to 30 minutes. Thereafter, the expression vectortreated in the manner described above was added to a medium containing1×10⁹ cells/l of FreeStyle 293F cells, and culture was conducted for 3days.

21-3. Purification and Preparation of Recombinant mFZD7c10-hFcm

21-3-1. Pretreatment of Culture Supernatant

After culture, the supernatant was recovered, filtered through a 0.22 μmfilter (TC Filter Unit, PES, Nalgene), and then cooled to 4° C. (in alow-temperature chamber). When cryopreserved, the resultant was thawedand then filtered through a 0.22 μm filter again.

21-3-2. Antibody Affinity Chromatography

The acidic buffer used is 1 liter of a solution comprising 3.895 g ofcitrate monohydrate (Nakalai Tesque, Inc., Japan, MW: 210.14), 0.38 g oftrisodium citrate (Wako Pure Chemical Industries, Ltd., MW: 258.07), and8.77 g of sodium chloride (Junsei Chemical Co., Ltd., MW: 58.44)dissolved in Milli-Q water. The neutralizing buffer used is 1 liter of asolution comprising 13.1 g of sodium dihydrogen phosphate dihydrate(Kanto Chemical Co., Inc., MW: 156.01), 41.5 g of disodium hydrogenphosphate dodecahydrate (Wako Pure Chemical Industries, Ltd., MW:358.14), and 8.77 g of sodium chloride (Junsei Chemical Co., Ltd., MW:58.44) dissolved in Milli-Q water.

The pretreated culture supernatant (1 liter) was applied to a protein Gcolumn (Hi Trap Protein G HP, 5 ml, GE Healthcare Bio-Sciences Corp.,Japan) equilibrated with PBS (Dulecco's phosphate buffered saline,SIGMA). Thereafter, the column was washed with 25 ml or more PBS, thecolumn was then washed with 25 ml or more buffer prepared by adding NaClto PBS to bring the NaCl concentration to 1.85 mol/l, and the column waswashed again with 30 ml of PBS. After the completion of the washingprocedure, 25 ml of acidic buffer was added to the column, and thetarget protein was recovered. The target protein was neutralizedimmediately after recovery with the use of a neutralizing buffer.AKTAexplorer 10s (GE Healthcare Bio-Sciences Corp, Japan) was used inthe separation and purification procedure. Endotoxin was removed beforeuse.

21-3-3. Preparation of Purified Authentic Sample

The buffer in the purified authentic sample obtained in Example 21-3-2was substituted with PBS using an ultrafilter membrane VIVASPIN20 10,000MWCO PES (Sartorius Stedim Japan K. K., Japan), and then concentrated.After the completion of the concentration and substitution procedure,the resultant was filtered through a 0.22 μm filter (Millex G V,Millipore, Japan). The concentration procedure was carried out in aclean bench to the extent possible. All the procedures conducted inExample 21-3 other than those conducted in a clean bench were carriedout in a low-temperature chamber (+4° C.) or on ice. The finalpurification product was subjected to SDS-PAGE (CBB staining), andmonomers were detected under reducing conditions. A proteinconcentration was determined by measuring a specific absorbance at 280nm (A280 nm) (E1%, 1 cm=10.5).

Example 22

Analysis of Mouse to which Recombinant mFZD7c10-hFcm had beenAdministered22-1. Administration of Recombinant mFZD7c10-hFcm

In order to evaluate the efficacy of the recombinant mFZD7c10-hFcm, therecombinant mFZD7c10-hFcm was administered to mice. Since therecombinant mFZD7c10-hFcm is a protein comprising the human antibody Fcregion, the possibility of suppressing the activity of the recombinantmFZD7c10-hFcm upon production of the neutralizing antibody in the bodyresulting from administration was considered. In order to reduce a riskof production of the neutralizing antibody, accordingly, the homozygote(the 97 KD mouse, CLEA Japan, Inc., Proc. Natl. Acad. Sci., U.S.A., 97:722-7, 2000) obtained via back-crossing of the immunoglobulin μ chaingene knockout mice lacking functional B lymphocytes and producing noantibodies into the MCH (ICR) strain (CLEA Japan, Inc.) was used for theadministration experiment. During the administration period, 97 KD micewere raised while humidity, temperature, and light conditions were keptconstant (temperature: 22° C.; humidity: 55%; 12 hours of light and 12hours of darkness) where they were allowed to freely eat feeds (CE-2,CLEA Japan, Inc.). Mice were divided into four groups (each groupconsisting of 5 mice) based on body weights at age of 6 weeks on theprevious day of the initiation of administration (i.e., day-1). Therecombinant mFZD7c10-hFcm was diluted with PBS to adjust the proteinconcentration to 5 mg/ml, and the resultant was cryopreserved, thecryopreserved product was thawed at the time of use, and the resultingsolution was administered through the caudal vein to the group of themFZD7c10-hFcm recombinant administration in amounts of 200 μl per mouseat 1 mg/dose once every 10 days (7 times in total) (q10d7). As a controlgroup, a non-treatment group was designated. The day of the initialadministration was designated as day 0, the recombinant protein wasadministered to the caudal vein every 10 days up to day 60 (seven timesin total), and all mice were subjected to necropsy on day 70.

22-2. Measurement of Bone Strength

The right femur samples were obtained at necropsy and subjected to athree-point bending test. When conducting a test, the span of thesupport points was set as 6 mm, and a load was applied at the midpointof the span to measure the maximum load (N).

As a result of measurement of the maximum load of femur samples obtainedfrom 5 control mice and 5 mice to which the mFZD7c10-hFcm recombinanthad been administered, the measured values were found to have increasedin the group of mice to which the mFZD7c10-hFcm recombinant had beenadministered (average: 28) compared with the values of the control group(average: 26). This indicates that the increased maximum load of thefemur may have been induced by administration of the mFZD7c10-hFcmrecombinant.

22-3. Analysis of Bone Structure of Mouse to which RecombinantmFZD7c10-hFcm had been Administered (3-Dimensional Microfocus X-Ray CT)

The left tibia samples were obtained at necropsy, and the internalstructure of the cancellous bone region of the proximal tibialmetaphysis was observed using a high-resolution microfocus X-ray CTscanner (micro-CT, Scan Xmate-L090, Comscantecno Co., Ltd., Japan) andthe analytic software (TRY 3D-BON, Ratoc System Engineering Co., Ltd.,Japan) in a non-invasive manner. The bone volume/tissue volume (BV/TV),the trabecular thickness (Tb. Th), the trabecular number (Tb. N), thetrabecular separation (Tb. Sp), and trabecular spacing (Tb. Spac) weremeasured (FIG. 25).

The internal structure of the cancellous bone of the tibia samplesobtained from 5 control mice and 5 mice to which the mFZD7c10-hFcmrecombinant had been administered was observed via micro CT. As aresult, the average bone volume/tissue volume, trabecular thickness, andtrabecular number were found to have increased, and the averagetrabecular separation and trabecular spacing were found to havedecreased in the group of mice to which the recombinant mFZD7c10-hFcmhad been administered compared with the control group. This suggeststhat the increased bone volume/tissue volume, the increased trabecularthickness, the increased trabecular number, the decreased trabecularseparation, and the decreased trabecular spacing in the cancellous boneof the proximal tibial metaphysis may have been induced byadministration of the recombinant mFZD7c10-hFcm (Table 31).

TABLE 31 Bone Trabecular Trabecular Trabecular Recombinant volume/tissueTrabecular number separation spacing administration/ volume thickness(Tb. N, (Tb. Sp, (Tb. Spac, no recombinant (BV/TV, %) (Tb. Th, μm) 1/mm)μm) μm) administration Average Average Average Average AveragemFZD7c10-hFcm 8.2/6.7 39.4/35.1 2/1.8 451.7/519.4 491.1/554.5 group vsnon-treatment group

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention can increase bone mass, bone density, and/or bonestrength. Accordingly, bone diseases resulting from osteoporosis,osteoarthritis, articular rheumatism, or malignant tumors, and variousdiseases and disorders associated therewith can be treated withoutcausing side effects.

SEQUENCE LISTING FREE TEXT

SEQ ID NOs: 3 and 4: Human IgG1 Fc mutantsSEQ ID NO: 5: DNA encoding a fusion proteinSEQ ID NO: 6: Fusion proteinSEQ ID NO: 9: DNA encoding a fusion proteinSEQ ID NO: 10: Fusion proteinSEQ ID NO: 13: DNA encoding a fusion proteinSEQ ID NO: 14: Fusion proteinSEQ ID NO: 17: DNA encoding a fusion proteinSEQ ID NO: 18: Fusion proteinSEQ ID NOs: 27 to 31: Fusion proteinsSEQ ID NOs: 38 to 43: DNAs encoding fusion proteinsSEQ ID NOs: 50 and 51: Sense oligo DNAs

SEQ ID NOs: 52 to 55: Primers

SEQ ID NO: 56: DNA encoding a fusion proteinSEQ ID NO: 57: Fusion protein

SEQ ID NOs: 62 and 63: Primers

SEQ ID NO: 64: DNA encoding a fusion proteinSEQ ID NO: 65: Fusion protein

SEQ ID NOs: 66 to 68: Primers

SEQ ID NO: 69: DNA encoding a fusion proteinSEQ ID NO: 70: Fusion protein

SEQ ID NO: 71: Primer

SEQ ID NO: 72: DNA encoding a fusion proteinSEQ ID NO: 73: Fusion protein

1-21. (canceled)
 22. A method for increasing bone mass, bone densityand/or bone strength, comprising; administering to a mammalian animalwith a disease selected from the group consisting of osteoporosis,osteoarthritis, articular rheumatism, hypercalcemia, Paget's disease ofbone, osteopetrosis, Camurati-engelmann's disease, arthropathy, primaryhyperthyreosis, osteopenia, osteohalisteresis, rachitis, traumatic bonefracture, and fatigue bone fracture, an effective amount of apharmaceutical composition which comprises, as an active ingredient, aprotein comprising an extracellular cystein-rich domain comprising theamino acid sequences of SEQ ID NO:21, 24 and 26, to increase bone mass,bone density and/or bone strength.
 23. The method according to claim 22,wherein the mammalian animal is a human.
 24. The method according toclaim 22, wherein the composition is simultaneously or continuouslyadministered in combination with another therapeutic agent forincreasing bone mass, bone density and/or bone strength.
 25. The methodaccording to claim 22, wherein the protein is a fusion protein of theextracellular cysteine-rich domain and a mammalian immunoglobulin Fcprotein or a mutant thereof prepared so that antibody dependent cellularcytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)activities are lowered.
 26. The method according to claim 22, whereinthe protein is chemically modified.
 27. The method according to claim26, wherein the chemical modification is a binding of one or morepolyethylene glycol molecules.
 28. The method according to claim 26,wherein the chemical modification is a binding of one or more sugarchains.
 29. The method according to claim 22, wherein the proteincomprises a fragment of an extracellular region of a human Frizzled 1, 2or 7 receptor, wherein said fragment comprises the extracellularcysteine-rich domain.
 30. The method according to claim 22, wherein theprotein is a recombinant protein.
 31. The method according to claim 22,wherein the extracellular cysteine-rich domain comprises an amino acidsequence spanning from the 1st cysteine residue on the N-terminal sideto the 10th cysteine residue in the amino acid sequence of anextracellular region of a human Frizzled 2 receptor.
 32. The methodaccording to claim 29, wherein the protein comprises the amino acidsequence of SEQ ID NO: 19, 22 or
 25. 33. The method according to claim25, wherein the Fc protein comprises the amino acid sequence of SEQ IDNO:
 4. 34. The method according to claim 25, wherein the nucleic acidencoding the Fc protein comprises the nucleotide sequence of SEQ ID NO:3.